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Structure-function relations in AMPA receptors

Arja KuusinenDepartment of Biosciences, Division of Biochemistry

Faculty of ScienceUniversity of Helsinki, Finland

Academic DissertationTo be presented for public criticism, with the permission of the Faculty of Sciences ofthe University of Helsinki, in the auditorium 2 of the Viikki Infocentre, Viikinkaari 11,

Helsinki, on March 24, 2000, at 12 o’clock noon.

Helsinki 2000

ISBN 951-45-9156-9 (PDF version)Helsingin yliopiston verkkojulkaisut

Helsinki 2000

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Supervised by:Professor Kari Keinänen

Department of Biosciences, Division of BiochemistryFaculty of Sciences and

Institute of BiotechnologyUniversity of Helsinki, Finland

Reviewed by:Professor Heikki Rauvala

Institute of Biotechnology andDepartment of Biosciences, Division of Biochemistry

Faculty of SciencesUniversity of Helsinki, Finland

and

Professor Mark JohnsonDepartment of Biochemistry and Pharmacy

Åbo Akademi, Finland, andTurku Center for Biotechnology

University of Turku, Finland

Opponent:Professor Esa Korpi

Department of Pharmacology and Clinical PharmacologyUniversity of Turku, Finland

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ORIGINAL PUBLICATIONS

This thesis is based on the following articles, which are referred to as I-IV in the text.

I. Kuusinen, A., Arvola, M., Oker-Blom, C. and Keinänen, K. (1995).Purification of recombinant GluR-D glutamate receptor produced in Sf21insect cells. Eur. J. Biochem. 233, 720-726.

II. Kuusinen, A., Arvola, M. and Keinänen, K. (1995). Molecular dissection ofthe agonist binding site of an AMPA receptor. The EMBO Journal 14, 6327-32.

III. Keinänen, K. Jouppila, A. and Kuusinen, A. (1998). Characterization of thekainate-binding domain of the glutamate receptor GluR-6 subunit. BiochemJ. 330, 1461-67.

IV. Kuusinen, A., Abele, R., Madden, D.R. and Keinänen, K. (1999).Oligomerization and ligand-binding properties of the ectodomain of the AMPAreceptor subunit GluRD. J. Biol. Chem. 274, 28937-43.

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TABLE OF CONTENTS

ABBREVIATIONS ------------------------------------------------------------- 5

ABSTRACT ---------------------------------------------------------------------- 6

1. INTRODUCTION ----------------------------------------------------------- 71.1. SYNAPTIC TRANSMISSION------------------------------------------------- 71.2. GLUTAMATE RECEPTORS IN MAMMALIAN CNS --------------------------- 71.2.1. Classification of glutamate receptors ------------------------------------ 91.2.2. Glutamate receptors and synaptic plasticity ---------------------------- 111.2.3. Pathophysiology of glutamate receptors -------------------------------- 141.3. MOLECULAR ANALYSIS OF IONOTROPIC GLUTAMATE RECEPTORS-------- 141.3.1.Subunit structure ------------------------------------------------------------ 151.3.1.1. Molecular cloning of the iGluRs --------------------------------------- 151.3.1.2. Primary structure -------------------------------------------------------- 171.3.1.3. Topology ------------------------------------------------------------------ 181.3.1.4. Modular structure of the iGluRs --------------------------------------- 191.3.2. Diversity of glutamate receptors ----------------------------------------- 241.3.2.1. mRNA editing ------------------------------------------------------------ 241.3.2.2. Alternative splicing------------------------------------------------------- 271.3.2.3. Post-translational modifications ---------------------------------------- 291.3.3. Assembly of glutamate receptors ---------------------------------------- 291.4. AIMS OF THE STUDY ------------------------------------------------------ 31

2. MATERIALS AND METHODS ----------------------------------------- 32

3. RESULTS ---------------------------------------------------------------------- 333.1. EXPRESSION OF RECOMBINANT NON-NMDA RECEPTORS------------------ 333.2. PURIFICATION OF RECOMBINANT AMPA RECEPTORS---------------------- 343.3. IDENTIFICATION OF THE GLUR LIGAND BINDING SITE-------------------- 353.4. DETERMINANTS FOR LIGAND SELECTIVITY ------------------------------- 373.5. BIOCHEMICAL CHARACTERISATION OF THE ECTODOMAIN --------------- 38

4. DISCUSSION----------------------------------------------------------------- 424.1. PURIFICATION OF A MEMBRANE BOUND CHANNEL PROTEIN ------------- 424.2. THE LIGAND BINDING SITE AS AN INDEPENDENT FRAGMENT ------------ 434.3. DETERMINANTS FOR LIGAND SELECTIVITY ------------------------------- 454.4. BIOCHEMICAL ANALYSIS OF OTHER EXTRACELLULAR DOMAINS --------- 454.5. CONCLUDING REMARKS --------------------------------------------------- 47

5. ACKNOWLEDGEMENTS ------------------------------------------------ 49

6. REFERENCES--------------------------------------------------------------- 50

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ABBREVIATIONS

AcNPV Autographa californica nuclear polyhedrosis virusAMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionateCaM-KII Ca2+- and calmodulin-dependent protein kinase IICNQX 6-cyano-7-nitroquinoxaline-2,3-dioneCNS central nervous systemDNQX 6,7-dinitroquinoxaline-2,3-dioneGABA γ-aminobutyric acidGluR glutamate receptor5-HT 5-hydroxytryptamine (serotonin)iGluR ionotropic glutamate receptorIMAC immobilised metal ion affinity chromatographyKBP kainate binding proteinLBD ligand binding domainLIVBP bacterial leucine/isoleucine/valine-binding proteinLAOBP bacterial lysine/arginine/ornithine-binding proteinLTP long-term potentiationmGluR metabotropic glutamate receptorMw molecular weightNMDA N-methyl-D-aspartateQBP glutamine binding proteinPAGE polyacrylamide gel electrophoresisPDZ PSD-95/Dlg/ZO-1PBP periplasmic binding proteinPKA cAMP-dependent protein kinasePKC protein kinase C

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ABSTRACT

The ionotropic glutamate receptors (iGluRs) are postsynaptic ion channelsinvolved in excitatory neurotransmission. The iGluRs can be classified according totheir specific agonists into N-methyl-D-aspartate (NMDA), α-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors. Each of these classescontains several homologous subunits that assemble in a subtype-specific mannerinto oligomeric (tetra/pentameric) complexes.

The iGluRs are integral membrane proteins for which there is as yet no structureavailable. However, some structural features of the subunits, including the primarystructure and its modifications, topology and domain organisation, have been solved.Extensive biochemical and biophysical characterisation has been so far hampered bythe lack of sufficient amounts of homogeneous material.

In this study, the production and purification of an AMPA-type glutamatereceptor was investigated. Recombinant GluRD receptors were expressed inSpodoptera frugiperda Sf21 insect cells and purified by affinity chromatography.The purified receptor preparation contained over 2000 pmol of high-affinity (K

d 52

nM) binding sites/mg protein and exhibited a single 110 kDa band on silver-stainedSDS-PAGE. A yield of 50-100 µg of purified receptor was obtained from one litre ofSf21 suspension culture.

In addition, the ligand binding sites of an AMPA receptor subunit GluRD anda kainate receptor subunit GluR6 were studied. Two discontinuous segments, S1 andS2, which show sequence similarity to bacterial amino acid binding proteins, wereexpressed as soluble secreted fusion proteins. The S1S2 fragment was shown tocomprise the ligand-binding domain in glutamate receptors as it reproduced the ligandbinding characteristics of an intact receptor. A role for the N-terminal third of the S2segment in AMPA-selectivity was identified by studying chimaeric GluRD/GluR6S1S2 fragments.

Moreover, the entire extracellular domain (XS1S2) and an N-terminal ~400residue segment (X) were expressed in High Five insect cells as soluble affinity-tagged recombinant proteins in order to study their structure and properties. The N-terminal X domain was shown not to contribute to ligand binding, but was suggestedto participate in the oligomerisation of the extracellular domain as a hydrodynamicanalysis of the domains showed dimerisation of the XS1S2 ectodomain but not of theS1S2 domain.

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1. INTRODUCTION

1.1. SYNAPTIC TRANSMISSION

The nervous system consists of neurons (1011 in human brain) and glial cells.Neurons are excitable cells; they generate and propagate electrical signals along theirprocesses forming networks that transmit and process information. Neuronscommunicate with each other at specialised junctions called synapses, originallyrealised by Ramon y Cajal in 1888 and demonstrated by electron microscopy in the1950s.

Chemical synapses can form between two neurons or a neuron and an effectorcell (e.g. a muscle cell or a secretory cell). The synapse involves a presynaptic nerveending, synaptic cleft and postsynaptic nerve ending (Fig. 1). In the presynaptic nerveterminus the neurotransmitter is stored in vesicles, which upon depolarisation of thenerve ending fuse to the plasma membrane in a Ca2+-dependent manner and releasethe transmitter into the synaptic cleft. The postsynaptic plasma membrane containsthe receptors for neurotransmitters, which are of two types: ligand-gated channelsand G-protein coupled receptors. In the former, binding of transmitter results in theopening of the channel and a resultant transmembrane ion flux. Transmitter bindingto a G-protein coupled receptor initiates a signal transduction cascade. Mostneurotransmitters are taken up from the synaptic cleft by the axon terminals or glialcells, but acetylcholine is degraded in the cleft by acetylcholine esterase.

Neurotransmitters are released locally from the synaptic terminal into thesynaptic cleft where they bind to and activate postsynaptic neurotransmitter receptors.Neurotransmission can be excitatory or inhibitory depending on the response of thepostsynaptic cell. The major excitatory transmitter in the central nervous system (CNS)is the amino acid L-glutamate. γ-Amino butyric acid (GABA) is the most prominentinhibitory transmitter in the forebrain, while glycine is the major inhibitory transmitterin the spinal cord and brainstem. Other neurotransmitters include acetylcholine,catecholamines, serotonin (5-HT), dopamine, histamine and ATP.

Virtually all neurons in the CNS respond to glutamate by depolarisation.Typically, responses at glutamatergic synapses exhibit two components (Fig. 2). Onesubclass of glutamate receptors called non-NMDA type glutamate receptors (GluRs)are responsible for the rapid onset and decay portion of the excitatory postsynapticcurrent (epsc), while another subclass known as NMDA receptors mediate thecomponent with slow rise time and decay (Westbrook and Jahr, 1989). Glutamateresponses are terminated by deactivation of receptors (dissociation of ligand from thebinding site) or desensitisation (closing of channel in the continued presence of agonist,detected only experimentally).

1.2. GLUTAMATE RECEPTORS IN MAMMALIAN CNS

Glutamate receptors have been implicated in many functions. They are themost important receptors in excitatory neurotransmission and subject to activity-dependent changes termed as “synaptic plasticity”, a mechanism suggested to underlie

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Figure 1. Schematic structure and function of a glutamatergic synapse. Upon depolarisation ofthe presynaptic nerve terminus, Ca2+ channels open and Ca2+ ions flow in. Fusion of glutamatecontaining vesicles is Ca-dependent. Vesicles release glutamate into the synaptic cleft whereit binds to glutamate receptors. Binding of glutamate to iGluRs opens cation channels causingdepolarisation of the membrane. Ca-flux via NMDA channels leads to activation of Ca-dependent enzymes and further signal transduction. From the synaptic cleft glutamate is takenup by glia cells, and finally returned to presynaptic terminus, where it is stored in vesicles.

Gln

Glu

astrocyte

presynaptic terminal

Ca2+

Gln Glu

Na+Na+, Ca2+

Glu

postsynaptic terminal

CaMK II

Cellular response

G-prot.mediatedpathways•PLC•cAMP

depolarisation

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Figure 2. Monosynaptic excitatory postsynaptic current (epsc) recorded from cultured CA1hippocampal neurons demonstrates the two receptor components of the glutamate-mediatedepsc. (Adapted from Westbrook and Jahr, 1989).

learning and memory (reviewed in Malenka and Nicoll, 1999). Glutamate receptorshave also a trophic role supporting the growth of new neurons (reviewed in Crair,1999). Furthermore, glutamate receptors have been implicated in pathologicalconditions (reviewed in Choi, 1988).

1.2.1. Classification of glutamate receptorsGlutamate receptors fall into two major structural categories; ionotropic

glutamate receptors (iGluRs) have an integral ion channel, whereas metabotropicglutamate receptors (mGluRs) are associated with G-proteins. The endogenousneurotransmitter L-glutamate activates all glutamate receptors, but the developmentof synthetic ligands as pharmacological tools has facilitated a further classification ofglutamate receptors (Fig. 3). Thus, iGluRs can be further classified as N-methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(AMPA) and kainate receptors according to their agonist affinities.

The AMPA receptors mediate the majority of all fast excitatoryneurotransmission. These receptors act with fast kinetics; the onset, offset anddesensitisation occur on a millisecond time scale (Jonas and Sakmann, 1992). Theendogenous agonist L-glutamate evokes currents carried mainly by the monovalentions Na+ and K+ and to a lesser extent by Ca2+ (Ascher and Nowak, 1988a). Theirspecific agonist, AMPA, binds with high affinity (with a K

d in the low nanomolar

range) and evokes currents exhibiting an initial fast desensitising component followedby a steady-state plateau similar to L-glutamate, whereas kainate activates non-desensitising currents (Sommer et al., 1990). Native AMPA receptors exhibit twotypes of channels, one showing low Ca2+ permeability and one with high Ca2+

permeability (Iino et al., 1990). Most native AMPA receptors exhibit low Ca2+

Figure 3. Structures of some glutamate receptor agonists. (Adapted from Armstrong et al.,1998).

permeability and linear current-voltage (I/V) relations (Mayer and Westbrook, 1987;Acher and Nowak, 1988b; Jonas and Sakmann, 1992). Minor subpopulations ofneurons such as GABAergic interneurons express Ca2+

-permeable AMPA receptorswith doubly rectifying I/V relations (reviewed in Jonas and Burnashev, 1995). Therectification is caused by cytoplasmic factors; endogenous polyamines (spermine,spermidine) block AMPA and kainate channels at resting membrane potentials in anactivity-dependent manner (Bowie and Mayer, 1995; Bowie et al., 1998).

Kainate receptors have similar fast activation and inactivation kinetics and asimilar affinity for glutamate as do the AMPA receptors (review by Lerma, 1997).The agonists L-glutamate, kainate, quisqualate and domoate elicit currents with inwardrectifying I/V relations and low Ca2+

permeability (Lerma et al., 1993). Kainate andAMPA receptors are often referred to as non-NMDA receptors, as they arepharmacologically not easily distinguished from one another.

In addition to glutamate, another endogenous agonist for NNIDA, receptors isaspartate, unlike in the case of non-NMDA receptors. The NMDA responses exhibitslower kinetics than the non-NMDA receptors, the current peaks approximately 10 msafter the non-NMDA receptor peak current and the depolarisation lasts for hundredsof milliseconds (Lester et al., 1990). NNIDA. receptors display two unique features:

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they require glycine as a coagonist (Johnson and Ascher, 1987; Kleckner andDingledine, 1988) and are blocked by Mg2+ ions in a voltage-dependent manner (Nowaket al., 1984; Mayer et al., 1984). Mg2+ ions block the current at normal negativemembrane potentials whilst at positive potentials they have almost no effect.Depolarisation of the membrane releases the block and conductance of the channelincreases. Like AMPA receptors, NMDA receptors show desensitisation in thecontinued presence of the agonist. However, they exhibit three kinds of desensitisationmechanisms depending upon the glycine and Ca2+ concentrations (Mayer et al., 1989;Sather et al., 1990). NMDA receptors are also regulated by a number of extracellularagents such as polyamines, Zn2+ and H+. Polyamines potentiate NMDA-mediatedcurrents by increasing the NMDA receptors mean open time (Lerma, 1992; Durand etal., 1992; Masuko et al., 1999). Zn2+, which is an abundant cation in excitatory terminalsand is released into the synaptic cleft during neurotransmission (Assaf and Chung,1984), inhibits currents mediated by NMDA receptors by binding to a site distinctfrom the Mg2+ binding site (Westbrook and Mayer, 1987). NMDA receptors are furtherregulated by the extracellular H+ concentration. They are inhibited almost completelyat pH5, and at physiological pH about half of the NMDARs are still inhibited (Tang etal., 1990; Traynelis and Cull-Candy, 1990).

The metabotropic glutamate receptors have no integral channel and theymediate slow, modulatory actions of glutamate in the nervous system by coupling,via GTP-binding proteins, either to inositol-1,4,5-trisphosphate (IP

3) formation and

intracellular Ca2+ mobilisation or to cAMP formation. There are three groups of mGluRsbased on their pharmacological properties and coupling to second messenger systems.Group I mGluRs activate type G

o/G

q family G proteins, which then stimulate

phospholipase C leading to hydrolysis of membrane phosphoinositides. Groups IIand III activate inhibitory G

i type G proteins, which inhibit adenylyl cyclase. The

mGluRs reside generally in the periphery of synaptic junctions, but some subtypesmay modulate glutamate release at presynaptic sites (Baude et al., 1993; reviewed inNakanishi, 1994).

1.2.2. Glutamate receptors and synaptic plasticityActivity-dependent modulation of neuronal connectivity is termed “synaptic

plasticity”. Synaptic plasticity is involved in memory acquisition, learning anddevelopment of the nervous system. It has been well-studied in hippocampal slices,where a laminar structure is maintained during slicing and cell layers are easilydistinquished. In an experimental model of long-term changes of synaptic strength,tetanic stimulation of input pathways in pyramidal neurons induces two temporally-distinct forms of synaptic potentiation: short-term potentiation (STP) that decays withinseconds to minutes and long-term potentiation (LTP) that can persist for days. LTPhas been suggested to be the synaptic basis of memory formation (reviewed in Blissand Collingridge, 1993).

LTP can be specifically blocked by NMDA receptor antagonists applied duringtetanic stimulation, thus indicating that NMDA receptors are critical in LTP induction.The previously discussed molecular properties of NMDA receptors, in particular thevoltage-dependent Mg2+ blockage and Ca2+ permeability, are suitable for its role as a

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“coincidence detector” in associative LTP, where association of synaptic inputs isneeded to depolarise the postsynaptic membrane sufficiently with simultaneouspresynaptic glutamate release.

There are two views on the location of the site of LTP expression; bothpresynaptic and postsynaptic mechanisms have been suggested. Regarding thepresynaptic mechanism, an increased probability of neurotransmitter release has beenreported (Malinov and Tsien, 1990; Bekkers and Stevens, 1990). An increase inpresynaptic activity in response to postsynaptic induction of LTP would require therelease of a diffusable messenger from the postsynaptic cell back into the synapticcleft. Arachidonic acid and nitric oxide have been suggested as candidate retrogradesignals (Bliss and Collingridge, 1993).

The postsynaptic mechanism of LTP would consist of an increased AMPAreceptor mediated response for which there are three possible mechanisms (Fig 4).First, the growth of new spines making new synapses in response to neuronal stimulationhas been suggested (Maletic-Savatic et al., 1999). Second, increasing amounts of AMPAreceptors in the postsynaptic membrane by delivery and clustering of receptors intospines from internal pools (Shi et al., 1999). A special form of this second possibilityoccurs during neuronal development when so-called “silent synapses” are activated(Durand et al., 1996). Silent synapses contain NMDA receptors, but lack AMPAreceptors and thus fail to respond to glutamate (Isaac et al., 1995; Liao et al., 1995).Insertion of AMPA receptors into the silent synapses enhances synaptic function (Shiet al., 1999). A third mechanism that can increase the postsynaptic response involvesmodulating the activity of existing synaptic receptors by covalent modifications such asphosphorylation, thereby allowing more ions to pass (Barria et al., 1997). It is probablethat all three mechanisms are used by neurons.

The molecular mechanisms leading to the aforementioned increase in synapticstrength have been studied intensively. A crucial event seems to be the NMDA receptormediated rise in the postsynaptic calcium concentration, which triggers a cascade ofbiochemical events (reviewed in Ghosh and Greenberg, 1995). Ca2+ binds to Ca2+-dependent enzymes, e.g. Ca2+-calmodulin dependent kinase (CaM-KII) therebyactivating them. This leads to activation of signalling pathways propagating the signalthrough to the nucleus where it activates transcription factors leading to synthesis ofnew proteins, which has been shown to be needed in order to convert STP into LTP(Casadio et al., 1999).

Long-term depression (LTD) is an opposite event to LTP characterised byuse dependent decrease in synaptic strength: weakening of synapses is induced bylow-frequency stimulation. Pathways involved in induction of LTD are complex andinvolve many of the same components as with LTP. NMDA receptor-mediated Ca2+-entry to postsynaptic cell is one mechanism, inducing intracellular events (e.g.dephosphorylation of sinalling proteins) leading to LTD. Contrary to LTP, expressionof LTD seems to involve removal of AMPA receptors from synapses (Luthi et al.,1999).

During development, glutamate receptors are involved in neuronaldifferentiation, migration and activity-dependent synapse formation. NMDA receptorshave been suggested to regulate activity-dependent neural circuit development. One

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Figure 4. Simplified model for the expression of LTP. Ca2+ ions flowing into the dendriticspine through NMDA receptors bind to calmodulin (CaM) to activate CaM kinase II byautophosphorylation. CaMKII phosphorylates AMPA receptors already present in thepostsynaptic membrane, thus increasing their channel conductance. It has also been suggestedthat CaMKII activity is involved in the insertion of more AMPA receptors into the postsynapticmembrane. (After Malenka and Nicoll, 1999).

Ca2+

CaM

CaMKII

AMPAR

P

CaMKII

P

AMPAR

AMPAR

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mechanism might be through expression of different NMDA receptors subunits, whichare expressed differently during development and have distinct ionic conductances(reviewed in Crair, 1999).

1.2.3. Pathophysiology of glutamate receptorsAll excitatory neurotransmitters are toxic to neurons in high concentrations

and/or when exposed for long periods, a condition termed excitotoxicity (reviewed inChoi, 1988; Lee et al., 1999; McNamara, 1999). Glutamate excitotoxicity has beenshown in animal models and cell culture, where AMPA and NMDA receptorantagonists provide protection from cell death and neuronal damage. This pathologicalcondition can occur in stroke, hypoxia and hypoglycemia via energy depletion resultingin the decreased uptake of synaptically released glutamate by glutamate transportersand accumulation of extracellular glutamate. The immediate effect in ischemic (resultingfrom deprivation of blood supply) trauma is the overactivation of iGluRs and Na+ andCl- flux into neurons, resulting in osmotic swelling and necrosis of cells. Secondarily,influx of calcium may trigger apoptotic pathways leading to further loss of neurons.

In addition to these acute insults, glutamate neurotoxicity can be a factor inseveral chronic neurological diseases. For example, epileptic seizures result from apersistent increase in neuronal excitability (reviewed in McNamara, 1999). Glutamatereceptors have been suggested also to have a role in degenerative diseases such asAlzheimer’s disease or Huntington’s disease, which feature gradual, selective loss ofneurons (reviewed in Choi, 1988).

1.3. MOLECULAR ANALYSIS OF IONOTROPIC GLUTAMATE RECEPTORS

Initially, the presence of glutamate binding sites in the brain was shown bydirect radioligand binding to membranes and ligand autoradiography of brain slices.NMDA and AMPA/kainate –binding sites have been solubilised and purified fromrat, pig and bovine brain (e.g. Henley and Barnard, 1989; Chang et al., 1991; Wentholdet al., 1992; Hall et al., 1992). The iGluRs in mammalian brain are of low abundanceand thus the material obtained in these experiments has been heterogeneous. However,these experiments have shown that the glutamate receptor is an oligomeric complex,which can be extracted in detergent solutions in an active form. In contrast tomammalian brain, high-affinity kainate binding sites are abundant in lower vertebrates.A kainate-binding protein (KBP), which shows pharmacological similarities tomammalian kainate receptors, has been purified to 90 % homogeneity from chickcerebellum by conventional fractionation (Gregor et al., 1988). Since 1989, cDNAsencoding subunits of iGluRs have become available, making it possible to expressthese proteins in heterologous systems and to obtain more detailed information ontheir molecular characteristics.

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1.3.1. Subunit structure1.3.1.1. Molecular cloning of the iGluR subunits

Ionotropic glutamate receptors are oligomeric proteins, which presumablyconsist of four or five homologous subunits. To date, 17 different subunits have beenidentified by molecular cloning, which correspond to the known pharmacologicalsubclasses of the glutamate receptors.

AMPA receptorsThe use of oocyte expression systems and functional cloning resulted in the

isolation of the first glutamate receptor cDNA clone (Hollmann et al., 1989). Injectionof in vitro transcripts from pools of rat brain cDNA-library into Xenopus oocytes andsubsequent analysis of kainate responses in the injected oocytes allowed subpoolingof the library until a single clone was obtained. This clone was named GluR-K1, as itwas originally regarded as a kainate receptor. At the same time cDNAs encoding twokainate-binding proteins from frog and chick were isolated and characterised (Wadaet al., 1989; Gregor et al., 1989). Subsequently, homology screening was used toisolate several further cDNA clones (Keinänen et al. 1990; Boulter et al., 1990;Nakanishi et al., 1990). The cloned subunits, termed alternatively as GluRA-D orGluR1-4, represented AMPA receptor subunits as confirmed by typical AMPAresponses and [3H]AMPA binding in cells expressing the cDNAs. AMPA receptorsubunits are able to form homomeric receptors, but native receptors are believed to bemainly heteromeric assemblies. In situ hybridisation revealed that the AMPA receptorsubunits are abundantly expressed in the brain in different layers of the cerebral cortex,caudate-putamen, hippocampus, cerebellum (in different cell layers) and olfactorybulb. GluRA and -B are also expressed in the hypothalamic nuclei and in amygdala(Keinänen et al., 1990).

The different AMPA receptor subunits have very similar pharmacologies,thus the targeted disruption of specific subunit genes has been employed to study thespecific functions of one AMPA receptor subunit, GluRB. A GluRB subunit knock-out results in impairment in the behaviour of GluRB-/- mice, indicating that the GluRBsubunit is critical for normal brain function (Jia et al., 1996).

Kainate receptorsCloning of the five members of the kainate receptor family was carried out

soon after the cloning of the AMPA receptor subunit. The kainate receptor subunitscan be grouped in two classes based on their affinities to kainate: the lower affinity(K

d 50-100 nM) kainate binding subunits GluR5-7 (Bettler et al., 1990; Egebjerg et

al., 1991; Bettler et al., 1992) and the high-affinity (Kd ~5 nM) kainate receptor subunits

KA-1 and KA-2 (Werner et al., 1991; Herb et al., 1992). GluR5-7 subunits are able tomake homomeric channels that respond to glutamate and kainate with rapidlydesensitising currents (Bettler et al 1992). In contrast, the KA1 and KA2 subunits arefunctional only when coexpressed with GluR5, GluR6 or GluR7, suggesting that theyexist only as heteromeric complexes in the CNS (Herb et al., 1992). Heteromerscontaining KA1 or KA2 generate fast and fully desensitising currents but, in addition

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to glutamate and kainate, they also respond to AMPA (Herb et al., 1992; Schiffer etal., 1997). GluR7 has a ten-fold lower affinity to glutamate than other AMPA andkainate receptors and, unlike other kainate receptor subunits, is insensitive to domoate.

GluR5 is mainly expressed during development, but lower levels are expressedalso in several regions of adult rat brain, including cingulate and piriform cortex, severalhypothalamic nuclei, amygdala and lateral septum (Bettler et al., 1990). GluR6 transcriptsare found at high levels in adult rat brain in the cerebellar and dentate granule celllayers, pyriform cortex, caudate-putamen and the hippocampal CA3 region (Egebjerget al., 1991). The GluR7 expression pattern overlaps with GluR6 expression; it isexpressed in inner cortical layers, hippocampal CA3 and dentate gyrus regions, reticularthalamic nucleus, mammillary bodies, pons and cerebellum (Bettler et al., 1992). Thedistribution of KA-1 and KA-2 overlaps with expression patterns of GluR6 and GluR7.KA-1 is restricted to hippocampal regions CA3 and dentate gyrus, whereas KA-2 ismore widely expressed in adult rat brain in all hippocampal regions, cerebellum, cerebralcortex, pyriform cortex and caudate-putamen (Herb et al., 1992).

Disrupting the GluR6 gene does not cause obvious physiological effects.Kainate receptor deficient GluR6-/- mice are healthy, and are less sensitive to theneurotoxin kainate, thus exhibiting reduced susceptibility to kainate-induced seizures(Mulle et al., 1998).

NMDA receptorsThe first NMDA receptor subunit, NR1, was isolated by expression cloning

using Xenopus oocytes (Moriyoshi et al., 1991), whilst NR2A-D (Monyer et al., 1992;Ishii et al., 1993) and NR3 subunit cDNAs (Ciabarra et al., 1995; Sucher et al., 1995)were isolated by homology screening. The NMDA receptor subunit NR1 is regardedas an obligatory subunit of the NMDA receptor, in contrast, NR2A-D are consideredas more modulatory subunits. NR1 is expressed in all parts of the brain and during alldevelopmental stages (Moriyoshi et al., 1991). NR2A is expressed after birth in theentire brain, but it is most prominent in the cerebral cortex, hippocampus, cerebellumand olfactory bulb. NR2B is widely expressed at embryonic stages, but becomesmore restricted in localisation after birth, with its main expression in the forebrain.NR2C is expressed postnatally in cerebellum, whilst NR2D is expressed at embryonicstages in the diencephalon and lower brainstem regions; its mRNA levels are stronglyreduced after birth (Ishii et al., 1993).

The roles of NMDA receptors were confirmed by studies on geneticallymodified mice. NR1-/- mice die perinatally due to respiratory failure, but show noobvious structural or histological defects in the CNS (Li et al., 1994; Forrest et al.,1994). NR2B-/- mice also die soon after birth presumably because they are not able tofeed (Kutsuwada et al., 1996). NR2A-/-, NR2C-/- and NR2D-/- mice are viable and shownormal brain morphology, but exhibit deficiencies in motor functions (Sakimura etal., 1995; Ikeda et al., 1995; Ebralidze et al., 1996; Kadotani et al., 1996). Thus, itseems that the NR1/NR2B subtype is vitally important, whereas other NR2 subunitsmay serve less crucial roles or are more easily compensated by other subunits.

The NR3 subunit is expressed in the adult rat brain in thalamus and spinalcord, but much higher levels of the mRNA transcript are detected in the developing

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brain (Ciabarra et al., 1995). NR3 shows rather low sequence similarity (around 27%identity) to other NMDA receptors and may function as a modulatory subunit; noindependent function has been assigned to it. NR3 subunit expression peaks at thesecond postnatal week and very little is expressed in the adult rodent brain. Therefore,NR3 has been suggested to have a role in neurite outgrowth and synapse formation(Sucher et al., 1995). This is supported by the observation that in NR3-/- mice themorphology of dendritic spines is modified (spine heads were enlarged and neckselongated) and their number is increased (Das et al., 1998).

Two further subunits having 27% sequence identity to other iGluRs wereidentified by homology screening of cDNA libraries and termed δ1 and δ2 (Lomeli etal., 1993). They do not respond to glutamate or any other ligands and accordinglythey are called “orphan” receptors. The δ2 subunit is selectively localised in thecerebellum and disruption of the δ2 gene in mouse produces a phenotype with impairedmotor coordination and reduced cerebellar long-term depression (LTD), which hasbeen suggested to be the cellular basis for motor learning (Kashiwabuchi et al., 1995).

The iGluR subunits in mammals are extremely well conserved between speciesshowing >95% amino acid identity between homologous subunits in rat, mouse andhuman (Puckett et al., 1991; Planelles-Cases et al., 1993). GluRs are also found inother vertebrates, e.g. fish (85% sequence identity between homologous subunits,Kung et al., 1996) and in invertebrates, but here the similarity is lower, e.g. less than50% amino acid identity between mollusc or insect and mammalian iGluRs (reviewedin Darlinson, 1992). Furthermore, Caenorhabditis elegans has glutamate receptors inthe inter- and motor neurons, which show closest sequence similarity to the AMPAreceptors (Maricq et al., 1995). Surprisingly, a family of iGluR subunits has beenidentified in the plant Arabidopsis thaliana. Preliminary results suggest that two ofthese proteins (GLR1 and GLR2) may be involved in light signalling (Lam et al.,1998). The two clones show sequence identity of less than 20% overall, but in certaindomains up to 60% with the mammalian glutamate receptors. Quite recently, aprokaryotic glutamate receptor, GluR0, was described from Synechocystis. GluR0shows amino acid sequence homology to both eukaryotic glutamate receptors andpotassium channels, and has thus been suggested to form a link between these two ionchannels (Chen et al., 1999).

1.3.1.2. Primary structureThe sequences of the ionotropic glutamate receptors code for polypeptides of

around 900 amino acids with apparent molecular mass of ~100 kDa, except for theNMDAR2 subunits, which have a large (350-600 aa) carboxy-terminal extension,and thus exhibit polypeptide lengths of 1250-1482 amino acids. The KBPs differfrom the other glutamate receptors in lacking the large N-terminal domain thus havingless than 500 aa with M

r ~49 kDa. The sequence identities within subfamilies are 50-

70% and between subfamilies 20-40%. Hydrophobicity analysis predicted that theionotropic glutamate receptor polypeptides have an N-terminal signal sequence andfour putative transmembrane segments (M1-M4), three closely spaced in the middleof the polypeptide and the fourth closer to the C-terminus. Several concensusglycosylation and phosphorylation sites are present in the subunit polypeptides.

18

Figure 5. Schematic presentation of the AMPA receptor subunit structure. The N-terminus ofthe receptor is on the extracellular side as well as the ligand-binding domain forming S1 andS2 segments. The C-terminus is intracellular. The subunit has three membrane traversingsegments and one re-entrant loop (M2).

X

S1 S2

extracellular

intracellular

M2

1.3.1.3. TopologyThe topology of a membrane protein defines the orientation of the protein

with respect to the plane of the membrane indicating which parts of the protein areextracellular and which are intracellular. Glutamate receptors have an N-terminalsignal sequence suggesting that the entire amino-terminal part until the firsttransmembrane segment is extracellular. This has been confirmed by a mutagenesisstudy showing that amino acids preceding M1 are involved in the binding of ligandand therefore must reside extracellularly (Uchino et al., 1992).

Hydropathy analysis of glutamate receptor sequences predicted an evennumber (four) of transmembrane domains. The simplest model deduced from thisprediction would place the carboxy-terminal part of the receptor subunit on theextracellular side together with the amino-terminus. This is, however, not consistentwith the experimental evidence. Ionotropic glutamate receptors are regulated byphosphorylation (Keller et al., 1992; McGlade-McCulloh et al., 1993). Several C-terminally located serine residues in NR1 were found to be phosphorylated and sincephosphorylation by PKA and PKC is an intracellular event, the C-terminus must beintracellular (Tingley et al., 1993).

19

As the N- and the C-termini reside on different sides of the membrane, theremust be an uneven number of transmembrane segments. The orientation of the largehydrophilic loop between M3 and M4 has been studied by glycosylation andphosphorylation site mapping. Initially, this loop was reported to be phosphorylatedat Ser684 (GluR6) and Ser696 (GluRB; Raymond et al., 1993; Wang et al., 1993;Nakazawa et al., 1995) implying that the loop is intracellular. On the other hand, Asn720in GluR6 is N-glycosylated and should therefore be extracellular (Roche et al. 1994;Taverna et al., 1994). The membrane topology of the homologous goldfish kainatebinding protein has been studied by deleting the M2 segment. If this was a truetransmembrane segment, then in the deletion mutant the rest of the polypeptide wouldhave an inverted topology. The goldfish KBP has a single N-glycosylation site in theM3-M4 loop, which is glycosylated irrespective of the presence of M2, indicating thatM2 is not a true transmembrane domain (Wo and Oswald, 1994; Wo and Oswald,1995a). Furthermore, new potential N-glycosylation sites were introduced into theGluR1 AMPA receptor subunit and their susceptibility to glycosylation was studied(Hollmann et al., 1994). This study showed that the loop between M3 and M4 residesextracellularly. Thus, apart from the contradictory phosphorylation studies most ofthe experimental evidence favors a model in which the termini are on opposite sidesof the membrane and M2 does not traverse the membrane, but rather forms a re-entrant loop (Fig. 5).

1.3.1.4. Modular structure of the iGluRsStructure-function studies for glutamate receptors became relevant after the

subunit sequences were obtained. Important problems to be addressed included thelocation of the agonist binding site, the ion channel and the structures which couplebinding to channel gating, as well as the location of allosteric regulatory elements.Sequence comparisons revealed two separate homologies to bacterial proteins whichbind amino acids (Nakanishi et al., 1990; O’Hara et al., 1993). The first is located inthe amino-terminal domain and the second is over the segments preceding M1 andbetween M3 and M4. This suggests a modular structure for glutamate receptors (Fig.5;reviewed in Wo and Oswald, 1995b), in which different parts may interact in anallosteric fashion. This modular character is also supported in the interchangeabilityof parts in chimaeric receptors (e.g. Stern-Bach et al., 1994; Villmann et al., 1997;Villmann et al., 1999).

The poreTo study the channel structure, the segments forming the channel needed to

be located; from the primary structure the likely location is in the hydrophobic segmentsM1-M4. Several pieces of evidence suggest that the M2 segment has a critical role inchannel formation. The sequence of the M2 segment is more hydrophilic than theother predicted transmembrane segments. It has also been shown to share sequencehomology to a K+ channel pore-loop domain, for which the structure has been determinedand shown to be a re-entrant loop (Bennet and Dingledine, 1995; Wood et al., 1995).The most convincing evidence for the role of M2 in forming the channel comes from

20

studies in which a single amino acid was found to be critical for ion permeability. Thisresidue is in the so-called Q/R site, which resides in M2 and is occupied by an arginineresidue in GluRB subunits, whereas in other AMPA receptors a glutamine residue isfound in this position (Verdoorn et al., 1991; Hume et al., 1991; Burnashev et al., 1992).The identity of the amino acid at this site determines the Ca2+-permeability andrectification observed in the current-voltage relation of AMPA mediated currents. Anarginine residue makes the channel Ca2+ impermeant with linear or inwardly rectifyingI/V relations, whereas a glutamine residue is responsible for Ca2+ permeant channelswith doubly rectifying I/V relationships. In the NMDA receptors an asparagine locatedat this site (therefore termed as the Q/R/N site) imparts to the receptor the voltage-dependent blockage by Mg2+ (Burnashev et al., 1992). Taken together, these resultssuggest that the M2 segment constitutes a critical part of the channel domain.

The M1 and M3 segments also contribute to the channel. The NMDA receptoropen channel blockers phencyclidine (PCP) and dizocilpine (MK-801) bind to residuesin the M2 and M3 segments. Mutational analysis identified PCP and MK-801 bindingsites, which involved residues in NR1: Asn-598 and Trp-593 in M2 and Ala-627 inM3 (Ferrer-Montiel et al., 1995). In the GluR6 kainate receptor, the M1 segment alsohas two sites critical for Ca2+-permeability, the so-called I/V and Y/C sites, which are,like the Q/R site in AMPA receptors, subject to RNA editing (Köhler et al., 1993; seechapter 1.3.2.1.). Hence, it seems likely that the transmembrane segments M1 andM3 contribute to the channel.

The structure of the M2 domain, previously thought to span the membrane,was studied by using engineered epitopes sensitive to proteolysis, together with nativeand introduced glycosylation sites. The structure of the pore was assessed by using thesubstituted cysteine accessibility method (SCAM, Kuner et al., 1996). The periodicityof the exposed residues is compatible with an α-helical secondary structure for themain part of M2 and an extended structure for its C-terminal part.

The channel is likely to consist structurally of two vestibules separated by anarrow constriction. The diameter of the pore has been estimated by measuring thepermeation of different ions through the NMDA receptor channel, and a value of 5.5Å has been reported (Villarroel et al., 1995). In the same study, the diameter of theouter and inner vestibules was determined to be around 7.3 Å. For homomeric AMPAand kainate receptors the channel constriction seems bigger, 7.6-7.8 Å in diameter(Burnashev, 1996); the Q/R site does not affect the pore size.

The outer vestibule of the NMDA receptors contains a binding site for thedivalent ions Ca2+ and Mg2+ (Premkumar and Auerbach, 1996). The endogenouspolyamines (PA) spermine and spermidine bind to two sites, one near the cytoplasmicside of the pore and the other in the outer vestibule (Bowie and Mayer, 1995; Washburnand Dingledine, 1996; Bowie et al., 1998).

The glutamate receptor channel structure is thus not yet known, but thestructures of two other membrane channels are available. The three dimensionalstructure of a bacterial tetrameric K+ channel homologous to mammalian channelshas been determined by X-ray crystallography. The channel is formed by a selectivityfilter surrounded by a cone of α-helices. The selectivity filter is a similar hairpin loop asin the glutamate receptors, but with an inverted orientation in the membrane (Doyle et

21

al., 1998). An entirely different channel structure is present in the nicotinic acetylcholinereceptor, consisting of five membrane traversing α-helices (Hucho et al., 1986; Imotoet al., 1986; Toyoshima and Unwin, 1988; Unwin, 1995).

Extracellular domainsTwo separate ~150 residue segments, termed S1 and S2 (Fig. 5) show

homology to the glutamine binding protein QBP and the lysine/arginine/ornithine–binding protein LAOBP (Nakanishi et al., 1990; O’Hara et al., 1993; Stern-Bach etal., 1994). The amino-terminal ~400 residue segment preceding S1 shows a moredistant similarity to the bacterial periplasmic leucine/isoleucine/valine–binding protein(LIVBP), leucine binding protein (LBP) and to the N-terminal segment of mGluRs(O’Hara et al., 1993).

The ligand-binding domain (LBD) of glutamate receptors has been shown toconsist of the S1 and S2 segments. With mutations based on sequence similarity toLAOBP and QBP, it was possible to identify amino acids in the NR1 subunit of theNMDA receptor that are important for activation by the co-agonist glycine (Kuryatovet al., 1994). Chimaeric proteins made between GluRC and GluR6, and GluRB andGluR6 were originally used to identify the crucial role of the S1 and S2 segments inagonist binding for AMPA and kainate receptors (Stern-Bach et al., 1994; Tygesen etal., 1995). After the studies by Stern-Bach et al. (1994) and Kuryatov et al. (1994), itwas directly demonstrated that S1 and S2 are sufficient for the ligand-binding activityof AMPA receptors and can be produced as a separate, soluble S1S2 fusion protein(this study). The LBD in AMPA receptors is monomeric when expressed as a solubleconstruct (Chen and Gouaux, 1997; this study) indicating that the ligand binding sitemay well reside within one subunit, in contrast to acetylcholine receptors, where theligand is thought to bind to a site formed between two subunits (Blount and Merlie,1989). NMDA receptors have two kinds of agonist binding sites; the NR1 subunitharbors the binding site for glycine, whereas the glutamate-binding site resides in theNR2 subunit (Kuryatov et al., 1994; Laube et al., 1997).

Molecular modeling based on sequence alignments together with site directedmutagenesis has been used to predict the residues involved in ligand binding (Kuryatovet al., 1994; Hirai et al., 1996; Paas et al., 1996; Laube et al., 1997; Lampinen et al.,1998). Key residues responsible for binding of ligand in AMPA receptors seem to beequivalent to those in the bacterial PBPs; in particular, ionic interactions between theα-carboxyl group and α-amino group of glutamate and oppositely-charged residuesin the receptor seem to stabilize the agonist in the binding site (Lampinen et al., 1998).

The crystal structure of the kainate-bound complex of GluRB LBD (Armstronget al., 1998) confirmed the close similarity between glutamate receptors and PBPspredicted by earlier studies. The structure of S1S2 resembles a clam shell with aslightly ellipsoidal (57Å × 43Å × 35Å dimensions) shape and the two lobes (calledlobes 1 and 2) have α/β secondary structure with 11 helices altogether. S1 and S2contribute to both lobes as the polypeptide chain crosses over between domains. Theligand binds in the interdomain crevice with mainly polar interactions mediated byhydrogen bonds and essential ionic interactions. In the S1S2 domain there are threecysteines, of which one is free and two form a disulfide bridge (Abele et al., 1998).

22

This disulfide bridge is conserved in ionotropic GluRs and is a target for redox modulationof NMDARs (Köhr et al., 1994; Sullivan et al., 1994). The disulfide bridge appears tostabilize the open, unliganded conformation of the ligand-binding domain.

The mechanism of ligand binding to PBPs has been studied in some detail. It isbelieved that the ligand first binds to lobe 1 with low affinity and subsequently establishesinteractions with lobe 2, which closes the domain and leads to high-affinity binding.This mechanism has been termed the “Venus flytrap model” according to the well-known botanical example (Sack et al., 1989). This mechanism has also been suggestedfor the glutamate receptor ligand-binding domain based on the structural homology(Mano et al., 1996). According to Mano et al., in GluRs the closure of the ligand-binding site would lead to the desensitized state of the channel. This model predictssubstantial movements of the lobes leading to closure of the binding site. Such dramaticmovements of the lobes were not, however, observed with GluRs, in studies usingsmall angle X-ray scattering to measure the radius of gyration in the presence andabsence of glutamate (Abele et al., 1999). Furthermore, the GluR LBD contains adisulfide bridge that is absent from the PBPs, giving further structural constraints.Therefore, either the movement does not occur or it is so subtle, that it is difficult todetect. Thus the ligand binding mechanism can not yet be established.

The N-terminal domain with ~400 amino acids (“X domain”, Fig. 5) comprisesaround one third of the receptor protein, but has until recently been lacking anyfunctional role. No effect on agonist binding properties has been detected when thisdomain has been swapped between different iGluRs or deleted (Stern-Bach et al.,1994; this study), but reports concerning its function in allosteric regulation havebeen published (e.g. Choi and Lipton, 1999; Masuko et al., 1999). In NMDA receptors,this domain probably has sites for binding of regulatory compounds such as zinc,spermine and the NMDAR antagonist ifenprodil. The voltage-independent zincinhibition of the NMDA receptor involves histidines His42 and His44 of the NR2Asubunit (Choi and Lipton, 1999). Ifenprodil, which selectively inhibits NR2B-containing NMDA receptors, binds to a site distinct from the glycine and glutamatesites in an activity-dependent manner. This site was located at least partly to theproximal part of the NR1 amino-terminus by studying site specific mutations to theN-terminal domain of NR1 (Masuko et al., 1999). Furthermore, the N-terminal domainof NR2 subunits has been suggested to play a role in the glycine-independentdesensitisation of NMDA receptors (Krupp et al., 1998; Villarroel et al., 1998). NMDAreceptors have several forms of desensitisation, of which glycine-independentdesensitisation occurs in the presence of a saturating concentration of the co-agonistglycine, but is independent of calcium influx.

The extracellular domain has also recently been proposed to have a role in theoligomerisation of the protein (Leuschner and Hoch, 1999; this study). Chimaericand truncated subunits assemble into oligomeric receptors if the N-terminalextracellular domain is present. Furthermore, the assembly is subtype specific, i.e.AMPA and kainate receptors do not co-assemble (Leuschner and Hoch, 1999).

In addition to the aforementioned roles, the extracellular domain may interactwith proteins in the synaptic cleft. Recently, a secreted protein called neuronal activity–regulated pentraxin (Narp) was shown to recruit AMPA receptors into large aggregates

23

by interacting with the extracellular domains of AMPA receptors. (O’Brien et al.,1999). Also, heparin, a sulfated glycosaminoglycan, has been suggested to interactwith AMPA receptors and modulate their single channel kinetics by prolonging themean open times (Sinnarajah et al., 1999).

Carboxy-terminal domainThe carboxy-terminal segment varies in size, constituting less than one-tenth

of the size of AMPA and kainate receptors as well as in NR1, while it formsapproximately one-third of the NR2 subunits. This segment is poorly conserved amongglutamate receptors and does not show obvious homology to other proteins. The C-terminal region of glutamate receptors is intracellular (Fig. 5) and serves to anchorthe protein in the postsynaptic density (PSD) and to the signalling machinery. Thisdomain is also important for modulation of the receptor function, as it undergoesalternative splicing and contains sites for phosphorylation.

Yeast two-hybrid screening has been used to identify interactions betweenthe cytoplasmic domain of GluRs and postsynaptic density proteins. For example,PSD-95 (called also SAP90) has been shown to interact with the C-terminal sevenamino acids containing a tSXV motif in the cytoplasmic tails of NR2 subunits (Kornauet al., 1995). PSD-95 harbors three so-called PDZ (PSD-95/Dlg/ZO-1) domains, whichinteract with the NMDA receptor, a Src homology (SH3) domain and a guanylatekinase (GK) homology domain. Several other proteins, which contain PDZ domains,have been shown to interact with other GluR subunits, including AMPA receptors.Glutamate receptor interacting proteins (GRIP 1 and 2, ABP) containing six to sevenPDZ-domains (Dong et al., 1997; Srivastava et al., 1998; Dong et al., 1999) and aprotein interacting with C kinase (PICK1, Xia et al., 1998), which has a single PDZdomain, have been shown to physically interact with AMPA receptors and induceclustering of AMPA receptors in excitatory synapses.

Kainate receptors are also associated with PDZ-domain harboring proteins(Garcia et al., 1998). GluR6 associates specifically with the PDZ1-domain of SAP90,SAP102, and SAP97 via its C-terminal ETMA sequence. GluR5 has a similar sequencemotif (ETVA) in its C-terminus, and may also interact with the SAP PDZ-domains.Interestingly, KA-2 seems to interact with the SH3- and GK-domains of SAP90 andSAP102 via its C-terminal PXXP-motif (Garcia et al., 1998).

The PDZ-domain containing proteins are capable of making multipleinteractions to form protein networks including cytoskeleton and signal transductionmolecules. The PDZ-domains can oligomerize in a hetero- or homomeric manner,but the PDZ-domain containing proteins of AMPA and NMDA receptor-interactingproteins do not bind to each other, which leads to separate clusters of AMPA andNMDA receptors (Srivastava et al., 1998). The NMDA receptors appear to be morefirmly anchored to the cytoskeleton, whereas the AMPA receptors seem to be in amore fluid context and thus may be more rapidly recruitable (Allison et al., 1998).Activation of silent glutamatergic synapses has been proposed to occur via GluRB/Cinteraction with GRIP/ABP leading to PKC-mediated signal transduction (Li, et al.,1999). This results in more AMPA receptors being inserted into the postsynaptic

24

membrane or activation of the already existing receptors by binding to PDZ-domaincontaining proteins.

The N-ethylmaleimide-sensitive fusion protein (NSF), a hexameric ATPasethat plays a central role in general membrane fusion events, interacts with AMPAreceptors GluRB and GluRDc thus modulating AMPA receptor function (Nishimuneet al., 1998; Osten et al., 1998; Song et al., 1998). NSF interaction does not involvePDZ-domains and the binding sites for GRIP and NSF in the C-terminal domain ofAMPA receptor subunits are overlapping and thus the two proteins may compete forassociation with AMPA receptors. This might have a role in the turnover andinternalisation of the receptors upon synaptic plasticity (Luthi et al., 1999).

The physiological importance of the PDZ-domains has been studied in vivoby constructing mice having C-terminal deletions in their iGluR subunits. Transgenicmice carrying C-terminally truncated NR2 subunits are phenotypically similar to micewith knock-out of the entire subunit (reviewed in Sprengel and Single, 1998). Theexpression of the subunit is at the same level as in wild type mice and the emergingchannels are indistinguishable from wild type NMDA channels, but the amounts offunctional synaptic NMDARs are decreased. Mice carrying the 2B∆C-terminustruncation die soon after birth. 2A∆C mice are viable but exhibit impaired synapticalplasticity and 2C∆C mice display deficits in motor coordination. A possible explanationfor this phenotypic similarity is the lack of a physical linkage to intracellularcomponents. 2B∆C has been reported to result also in hindering of efficient clusteringand synaptic localisation of NMDA receptors (Mori et al., 1998).

In conclusion, intracellular interactions may provide a basis for the structuraland functional regulation of GluR expression; GluRs can be targeted to synapses in acell-type, developmental and use-dependent manner.

1.3.2. Diversity of glutamate receptors.Assembly of glutamate receptor subunits into various combinations creates a

variety of functionally and structurally different glutamate receptors. Further diversityis generated at the level of single glutamate receptor subunits by genetic mechanismsand post-translational modifications. Genetical mechanisms include two steps in theprocessing of pre-mRNA: RNA editing and alternative splicing, whereas proteinmodification includes the addition of N-glycans, phosphorylation and palmitoylation.

1.3.2.1. mRNA editingRNA editing is a newly identified genetic mechanism for changing gene-

specified codons and hence protein structure and function. In mammalian nuclearmRNAs it was first found in intestinal apolipoprotein B mRNA (Powell et al, 1987).Later, it was recognised in the serotonin-2C receptor (Burns et al., 1997) and K+-channel (Patton et al., 1997) mRNAs. However, it is best documented and characterisedin glutamate receptors (Fig. 6A; reviewed in Seeburg et al., 1998). Sequencing of theAMPA receptor subunit genes revealed that the arginine in the Q/R-site of GluRB isnot encoded genomically, but it is introduced by editing of a single nucleotide in theprecursor mRNA (Sommer et al., 1991). This Q/R site editing occurs also in the kainatereceptor subunits GluR5 and –6, but not in the other AMPA or kainate receptor subunits.

25

A.

B.

AMPAR

KaiR

R/GQ/RY/CI/V

KaiR

GluRAGluRBGluRCGluRD

GluR7

GluR5GluR6

AMPAR

GluRA

GluRB-short

GluRCGluRDcGluRD

GluRB-long

GluR7a

GluR5c

GluR6-1

GluR7b

GluR6-2

GluR5-2GluR5aGluR5bGluR5-1d

NMDAR

NR1-1aNR1-1b

NR1-2a

NR1-2b

NR1-3aNR1-3b

NR1-4a

NR1-4b

Figure 6. Editing and alternative splicing of iGluR subunits. The membrane-embeddedsegments are shown by blue boxes A. The editing sites are indicated for AMPA and kainatereceptor subunits. The NMDA receptor subunit mRNAs have not been found to be edited. B.The alternatively spliced cassettes are shown for different iGluR subunits by the differentiallycoloured boxes. (After Dingledine et al., 1999).

26

The editing of this site is controlled in a cell-type specific and developmental manner: inthe fetal rat brain both unedited and edited GluR-B forms exist, but at birth the GluRBsubunit is almost completely edited (99.9%, Burnashev et al., 1992). The GluR5 and 6subunits seem to be less efficiently edited than GluRB (Köhler et al., 1993). The editingdetermines the single-channel conductances of GluR5 and GluR6 channels so that theedited versions exhibit 25- and 10-fold reduced conductances as compared to theunedited GluR6 and GluR5, respectively (Swanson et al., 1996). In heteromeric kainatereceptors consisting of edited versions of either GluR5 or GluR6 and a KA-2 subunit(harboring a glutamine at the Q/R site), the single-channel conductances are higherthan in the homomeric channels. GluRB editing also reduces single-channel conductancein comparision to unedited GluRB (Swanson et al., 1997). Thus, the number of arginineresidues introduced by editing seems to determine the single-channel conductance.Interestingly, the NR3A subunit of NMDA receptors is the only subunit having anunedited arginine at the Q/R/N-site, i.e. an arginine is encoded genomically (Ciabarraet al., 1995).

Two other editing sites were found in the M1 segment of GluR6: isoleucineor valine at the I/V-site and tyrosine or cysteine at the Y/C-site (Köhler et al., 1993).Thus, eight isoforms of GluR6 may exist: the genetically coded QIY (10% of rat brainmRNAs), the fully edited RVC (comprising 65%) and six partially edited forms (25%of total GluR6 mRNAs). The M1 editing also contributes to the Ca2+ -permeability ofthe channel together with the Q/R site editing. Interestingly, it was found that if TM1is fully edited, then the arginine at the Q/R site confers higher Ca2+-permeability thanglutamine does, in contrast to the AMPA receptors.

Yet another editing position was found in the extracellular S2-segment ofAMPA receptors; the mRNAs of subunits GluRB, -C and –D are edited, introducinga glycine codon instead of an arginine codon (the R/G site, Lomeli et al., 1994). Theextent of editing is controlled in a cell-type and splice variant (see 1.3.2.2.) specificmanner and according to the developmental stage. The AMPA receptor channels editedat the R/G site exhibit faster recovery from desensitisation and thus larger steady-state currents too (Lomeli et al., 1994).

The mechanism of the glutamate receptor RNA editing appears to be via siteselective deamination of adenosine to inosine, occuring in the nucleus at the pre-mRNA stage. The intronic sequences are vital for Q/R site editing. The editing enzymerecognises a short double stranded RNA structure formed between an exonic sequencearound the editing site and a complementary sequence (ECS) in the downstream intron(Higuchi et al., 1993; Egebjerg, 1994; Herb et al., 1996). Several editing enzymes arenow identified; these are called adenosine deaminases acting on RNA (ADAR 1-3;reviewed by Bass et al. 1997). These enzymes are expressed in many tissues, and thusthe mechanism may be more widespread than originally anticipated.

RNA editing affects the channel properties of GluRs, and may therefore havean important physiological role. This has been studied by making transgenic and aknock-out mice incapable of editing GluRB subunits (Brusa et al., 1995; Feldmeyeret al., 1999). Heterozygous mice harboring an editing-incompetent allele of GluRBexpress unedited GluRB subunits and thus AMPA receptors with increased Ca2+-permeability. These mice show epileptical seizures and die before postnatal day 20.

27

Conversion of the high Ca2+-permeability exhibiting receptors into a low Ca2+-permeability species at birth seems thus to be critical for expression of healthyphenotype.

1.3.2.2. Alternative splicingGlutamate receptor subunits exist in different molecular versions arising from

alternative splicing of pre-mRNAs. Splicing has been shown to occur in the N-terminaldomain, C-terminal domain and in the extracellular loop between M3 and M4depending on the subunit (Fig. 6B). These splice variants add to the diversity of GluRsby showing different functional properties and expression patterns.

In all AMPA receptor subunits, a module called “flip-flop” can be chosenfrom two adjacent exons (exons 14 and 15) separated by an intron of ~900 bp (Sommeret al., 1990). This 115 bp module encodes a 38 amino acid segment in the S2-domain.The flip and flop cassettes are very similar, differing only in 9-11 residues. However,the alternative flip and flop versions of the AMPA receptor subunits have distinctexpression patterns and different pharmacological and kinetic properties. The twosplice variants differ in the distribution and expression levels in rat brain. In thehippocampus the cell specific nature of expression is clear; the flip form is the onlyisoform in the pyramidal cells in the CA3 area, whereas in the CA1 area both formscoexist (Sommer et al., 1990). The expression of flip- and flop-variants are alsodevelopmentally controlled. Early in development, before postnatal day 8 the flipform is the only form expressed, but after this stage the expression of the flop isoformincreases throughout the entire rat brain. By postnatal day P15 the adult-specific patternis established (Monyer et al., 1991).

The different functional characteristics of the flip and flop variants suggestthat splicing has a physiological role. The flip forms show slower desensitisation in Cand D subunits, but there is no difference in A and B subunits (Mosbacher et al.,1994). Studies with modulatory drugs have shown that the flip and flop domainsdiffer in their pharmacological properties. Allosteric potentiators of AMPA receptors,cyclothiazide and 4-[2-(phenylsulfonylamino)-ethylthio]-2,6-difluoro-phenoxy-acetamide (PEPA), have opposing effects on the flip and flop isoforms of GluRAreceptors. The flip form is more potentiated by cyclothiazide that the flop form (Partinet al., 1993). This difference was traced to a single residue, Ser750 on GluRA

i, which

is replaced by asparagine in the corresponding site in GluRAo (Partin et al., 1995 and

1996). Flop-forms, in contrast, are preferentially modulated by PEPA (Sekiguchi etal., 1997).

In the X-ray structure of GluRB LBD, the flip/flop domain forms two adjacentα-helices on the surface of the LBD (Armstrong et al., 1998). This suggests that itmay participate in the allosteric regulation of the channel by cyclothiazide and PEPAby coupling the movements of the LBD upon binding of ligand to the opening of thechannel, possibly by mediating this signal to the transmembrane domains.

AMPA receptor subunits GluRB and D are further varied via alternativesplicing of mRNAs producing two different C-termini (Köhler et al., 1994; Gallo etal., 1992). These are produced by insertion of a module 14 amino acids downstreamfrom M4. The longer GluRB C-terminus shows similarity to the C-termini of GluRA

28

and the longer GluRD. The shorter C-terminus of GluRB, on the other hand, is similarto those of GluRC and the shorter GluRD. Curiously, with GluRA and C no splicevariants have been found. Expression of different splice variants varies across the ratbrain as studied by in situ hybridisation. The majority of native GluRD subunits carrythe longer C-terminus, whereas only a minor fraction of GluRB subunits contain thelonger C-terminus.

Kainate receptor mRNAs also undergo alternative splicing. In GluR5 aninsertion of 15 residues in the N-terminal domain generates two splice forms, GluR5-1 and GluR5-2 (Bettler et al., 1990). In addition, GluR5 has three C-terminal splicevariants, which do not differ pharmacologically, but their expression levels are affectedin transfected cells (Sommer, 1992). Two GluR7 variants, known as 7a and 7b, divergeafter 14 amino acids downstream from M4 with a 40 nucleotide insertion resulting inalternative C-termini (Schiffer, 1997). GluR7b C-terminus shows no homology toother GluR sequences and exhibits smaller glutamate activated currents than 7a(Schiffer, 1997).

In the NMDA receptor subunit NR1, three exons undergo alternative splicing.Eight isoforms of NR1 are produced by combinatorial usage of three cassettes termedN, C1 and C2 (Sugihara et al., 1992; Hollmann et al., 1993). The N-cassette encodes21 amino acids that can be inserted in the N-terminal domain at residue 190. C1 andC2 (37 and 38 amino acids, respectively) are coded by adjacent exons and can beinserted into the C-terminal domain proximal to M4. Splicing-out of the segment thatencodes the C2 cassette removes the first stop codon and results in a new open readingframe that encodes an unrelated 22-residue segment, C2’. The splice variant expressionpattern reveals regional differences that do not change in the course of developmentdespite changes in abundance (Laurie and Seeburg, 1994). The variants lacking the N-cassette (a-variants) are uniformly expressed in rat brain, whilst the variants containingthe N-cassette (b-variants) are more restricted in expression. In primates the expressionpattern is similar (Meoni et al., 1998).

The NR1 a- and b-isoforms are also functionally different. The b-variantsexhibit larger current amplitudes and lower affinity for agonists, but greater affinity forantagonists than the a-variants (Sugihara et al., 1992; Durand et al., 1992; Hollmann etal., 1993). Low concentration of Zn2+ potentiates glutamate-gated currents at a-typechannels, but higher concentrations of Zn2+ are inhibitory to both a-and b-type channels(Hollmann et al., 1993). The NR1 N-terminal splice variants also have different pH-sensitivities; insertion of the N-cassette confers on the receptor lower sensitivity toextracellular protons (Traynelis et al., 1995). A similar effect is seen with polyaminesas they attenuate proton inhibition suggesting that the N-cassette might modulateNMDA receptor pH-sensitivity by interacting or shielding the extracellularly locatedpH-sensor. The splicing of the C-terminal domain regulates surface expression of thefunctional NMDA receptors (Ehlers et al; 1995; Okabe et al., 1999).

29

1.3.2.3. Post-translational modificationsGlycosylation is a post-translational modification whereby carbohydrate chains

are covalently attached to serines (O-linked glycosylation) or asparagines (N-linkedglycosylation) of the proteins. Addition of glycans is part of the protein’s maturationprocess, but its physiological functions are still poorly known. Glutamate receptorsharbor several potential sites for N-linked glycosylation in their extracellular domainsand several of these sites have been shown to be glycosylated in recombinantlyexpressed glutamate receptor subunits (Roche et al., 1994; Taverna et al., 1994).Intracellularly-located glutamate receptors have been reported to have simple, high-mannose type oligosaccharides, thus representing an immature pool of receptors,whereas the synaptic GluRs have complex, fully-processed sugar chains representingthe mature forms of receptors (Standley et al., 1998). The possible role of theasparagine-linked glycans in receptor function is not clear at present. Glycans are notnecessary for the ligand binding activity as shown by native-like ligand binding activityof the soluble AMPA receptor ligand binding domain expressed in Escherichia coli,an organism not capable of glycosylation (Arvola and Keinänen, 1996). However,treating cells with lectins such as concanavalin A (Con A), which binds specificallyto certain carbohydrates, potentiates currents elicited by glutamate, quisqualate andAMPA possibly by reducing desensitisation, suggesting that the glycans have afunctional role (Mayer and Vyklicky, 1989). N-glycosylation has been shown to bean absolute prerequisite for lectin–mediated inhibition of desensitisation, but thespecific site of lectin-binding does not appear to be important (Everts et al., 1997;Everts et al., 1999).

The phosphorylation state seems to be an important modulator of glutamatereceptor activity. The alternatively spliced C-termini of glutamate receptors differ bythe presence of phosphorylation sites (Tingley et al., 1993; Barria et al., 1997; Grantet al., 1998; Carvalho et al., 1999). Protein kinase C (PKC), cyclic AMP-dependentprotein kinase (PKA) and Ca2+-calmodulin dependent protein kinase II (CaM-KII)have been reported to phosphorylate serine and threonine residues in GluR subunitsin a sequence context-dependent manner. Phosphorylation has been observed afterinduction of LTP, and CaM-KII activity has been shown to be necessary and sufficientfor LTP (Barria et al., 1997, Pettit et al., 1994).

Palmitoylation, the covalent addition of fatty acid chains to a serine, threonineor cysteine residue in proteins via an ester or thioester bond, is one well-characterisedpost-translational modification, which also occurs in glutamate receptors. In GluR6,palmitoylation occurs at two cysteines (C827 and C840) located in the C-terminaldomain, but this modification does not change the kainate-gated currents (Pickeringet al., 1995). However, an unpalmitoylated mutant GluR6 was reported to be a bettersubstrate for protein kinase C than the wild type palmitoylated receptor, suggestingthat palmitoylation may indirectly modulate receptor function.

1.3.3. Assembly of glutamate receptorsGlutamate receptors, like other known ion channels, are oligomeric proteins

consisting of homologous subunits. Whilst the subunit composition of GluRs can

30

vary with cell type and stage of development leading to different functional properties,the subunits always assemble subtype specifically. Thus, e.g. AMPA receptor subunitscan co-assemble from different combinations of GluRA-D subunits, but they do notmake complexes with the kainate or NMDA receptor subunits (Partin et al., 1993;Puchalski et al., 1994).

The number of subunits is considered to be a well-conserved property within agiven channel protein family. Thus, voltage-gated potassium channels are known to betetramers, whilst muscle nicotinic acetylcholine receptors and other members of theacetylcholine receptor superfamily are pentameric (Doyle et al., 1998; Unwin 1995).Currently, the actual number of subunits in a multimeric glutamate receptor and thestoichiometry (the relative proportions of each subtype) is not known, nor have thedeterminants of assembly been identified. Being neurotransmitter receptors, glutamatereceptors were initially thought to structurally resemble the pentameric acetylcholinereceptors. The superficial similarity between the pore structures of tetrameric potassiumchannels and iGluRs suggests, however, that iGluRs might be composed of four subunitsinstead. Biochemical analysis of native receptors by using either hydrodynamic analysesor cross-linking are consistent with tetra- or pentameric assemblies for NMDA andAMPA receptors (Wenthold et al., 1992, Blackstone et al., 1992, Brose et al., 1993,Wu and Chang, 1994, Wu et al., 1996). Using native brain in cross-linking experimentsmaterial is problematic, however, because interacting proteins may co-purify with thereceptors. Recently, the stoichiometry of recombinant and native NMDA receptorshas been studied by successive immunoaffinity purifications. The receptor was suggestedto contain at least three NR2 subunits and two NR1 subunits, thus having a pentamericstructure (Hawkins et al., 1999).

Furthermore, functional studies of the receptor have been carried out to findout the number of subunits forming an ion channel. Modeling of single channel currentpatterns (Behe et al, 1995, Prenkumar and Auerbach, 1998, Rosenmund et al., 1998)and whole cell currents in hybrid receptors consisting of various proportions of mutantand wild type subunits (Ferrer-Montiel and Montal, 1996, Laube et al., 1998, Mano andTeichberg, 1998) have been employed. Assuming that two glutamate molecules areneeded for channel activation (Clements et al. 1998; Rosenmund et al. 1998) and thatsingle channel currents become larger when more glutamate is bound, it can becalculated how many binding sites are occupied (Rosenmund et al., 1998). However,the results obtained with these methods also segregate into two classes suggestingtetra- or pentameric assemblies.

31

1.4. AIMS OF THE STUDY

The aim of this study was to characterise the structure-function relationships in AMPAreceptor subunit GluRD and in kainate receptor subunit GluR6 in order to gaininformation on the molecular structure of ionotropic glutamate receptors and to establishmethods for direct structural analysis of iGluRs.

The specific goals of the study were1) to develop a method for efficient purification of recombinant glutamate receptors2) to identify and analyse the ligand-binding site in AMPA and kainate receptors3) to study the properties of the ectodomain of GluRD AMPA receptor

32

2. MATERIALS AND METHODS

The materials and methods are described in the original publications andtherefore only a list of methods is provided here.

Method Original publicationBaculovirus expression system I-IVChemical cross-linking IVFluorescence titration IVGel filtration chromatography IVImmobilised metal ion affinityChromatography -intact GluRD

I

Immobilised metal ion affinityChromatography -soluble domains

IV

Immunoaffinity chromatography-membrane proteins

I

Immunoaffinity chromatography-soluble proteins

II

Immunoprecipitation IVInsect cell culture I, IIProtein assay, BCA IProtein assay, amido black IRadioligand binding assay[3H]AMPA

I-IV

Radioligand binding assay[3H]Kainate

III

Recombinant DNA I-IVSDS-PAGE ISolubilisation of GluRD IStopped-flow kinetics IVSucrose density gradient centrifugation IVWestern blotting I

33

3. RESULTS

3.1. EXPRESSION OF RECOMBINANT NON-NMDA RECEPTORS (I AND III )

Glutamate receptors are not abundant in native nervous tissue, thereforerecombinant expression is necessary to produce sufficient amounts of homogenousmaterial for detailed biochemical, biophysical and structural analyses. In the presentstudy, homomeric GluRs, consisting of an AMPA selective GluRD subunit or a kainateselective GluR6 subunit, were expressed in insect cells providing a source ofhomogeneous material with a defined subunit composition.

Recombinant baculoviruses were prepared for expression of GluRD or GluR6.The constructs were engineered to harbor an N-terminal FLAG tag and a C-terminalHis tag to facilitate identification and purification. Sf21 insect cells were infected, inspinner flasks, with the recombinant viruses and cells harvested 3-4 d post-infection.Membranes from Sf21 cells infected with GluRD were prepared and tested forexpression of the receptor protein by SDS-PAGE followed by immunoblotting.Detection with α-FLAG tag antibody showed a 110 kDa band representing the GluRDsubunit, with no visible degradation products (IV , Fig. 2A, lane 1), whilst uninfectedcontrol cells or cells infected with wt AcNP virus showed no staining (not shown).The immunopositive samples also showed specific binding of radioligand [3H]AMPA.Characterisation of the binding properties revealed high-affinity [3H]AMPA-binding(K

d = 40 nM, I ) for membranes of GluRD-infected cells. In ligand competition assays,

the membranes showed typical AMPA selective pharmacology; the 50% inhibitoryconcentrations (IC

50) for agonists were 61 nM for AMPA, 390 nM for L-Glu and 2.2

µM for kainate. NMDA did not displace [3H]AMPA binding. The expression levelwas 2-6 nmol (0.2-0.6 mg) from a liter of insect cell culture. The properties (affinityand expression level) obtained here were similar to those obtained previously for theuntagged GluRD expressed in insect cells (Keinänen et al., 1994).

A kainate receptor GluR6 with an N-terminal FLAG-tag and a C-terminalHis-tag was also expressed in Sf21 cells. The membranes prepared from these cellsexpressed a 100-112 kDa broad band typical for glycoproteins, with a smaller, ~50kDa band possibly representing a degradation product (III, Fig. 3B, lane 1). Themembrane preparation bound [3H]kainate with high affinity (K

d = 18 nM). In ligand

competition assays, the agonists domoate (IC50

= 13 nM) and L-glutamate (IC50

= 0.6µM) and the antagonist CNQX (IC

50 = 3.8 µM) inhibited binding, whereas AMPA

and NMDA did not. These binding properties were similar to those described earlierfor mammalian cell-expressed GluR6 (Egebjerg et al., 1991; Sommer et al., 1992).

Hence, it is feasible to produce recombinant glutamate receptors in insectcells for structural studies. The recombinant receptors resemble the wild type receptorsexpressed in mammalian cells.

34

3.2. PURIFICATION OF RECOMBINANT AMPA RECEPTORS (I )

To obtain material for structural studies of AMPA receptors, solubilisationand purification of recombinantly expressed homomeric GluRD receptors were studied.

Solubilisation of insect cell membranes expressing GluRD harboring an N-terminal FLAG tag and a C-terminal His tag was studied using different detergents.Glycerol and the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) were includedin the solubilisation buffer for better preservation of the activity of the protein. After anovernight solubilisation at +40C, the mixture was clarified by high speed (160 000 × g)centrifugation for one hour to get rid of all insoluble material. The efficiency ofsolubilisation was followed by measuring [3H]AMPA binding activity in the supernatantbefore and after adding the detergent. The non-ionic detergents Triton X-100, TritonX-114, n-octyl-D-glucoside and n-dodecylmaltoside were most efficient in solubilisingGluRD. The concentration dependence of GluRD solubilisation in Triton X-100 wastested and 1% of the detergent was found to be sufficient, with no further solubilisationobtained with higher concentrations of Triton X-100. Under optimal conditions, 60-70% of the [3H]AMPA binding activity was solubilised with 1% Triton X-100.Solubilisation did not affect the ligand binding properties.

Purification was carried out using a two-step protocol. As the first step,immobilised metal ion affinity chromatography (IMAC) was employed. In preliminaryexperiments, several divalent ions (Ni2+, Zn2+, Co2+, Cu2+) were tested for binding ofsolubilised GluRD and best results were obtained with Ni2+. The bound receptorswere eluted from Ni2+ loaded Sepharose by using a rising imidazole step gradient. Atimidazole concentrations under 50 mM most of the nonspecifically or weakly boundcontaminating proteins were eluted, but the His-tagged GluRD eluted at 100 mMimidazole, as judged by immunoblots and [3H]AMPA binding assay. As a secondpurification step, an immunoaffinity column with monoclonal antibody M1 againstthe FLAG tag was used. The recognition of the epitope is Ca2+-dependent, and thusmild conditions for elution can be achieved by using EDTA. Pooled imidazole eluatesfrom IMAC were subjected to M1 immunaffinity chromatography in the presence of1 mM Ca2+. The receptor was eluted with 2 mM EDTA. In a silver-stained protein gel,a major 110 kDa band representing the purified GluRD was seen with a very faintband of ~70 kDa representing contaminants or breakdown products (I , Fig. 6B).

Treatment with N-glycosidase F reduced the size of the purified protein from110 kDa to 100 kDa indicating that purified GluRD is N-glycosylated. The purifiedGluRD showed high-affinity [3H]AMPA binding (K

d = 40-60 nM) and the ligand

binding characteristics were unaltered by purification. The preparations showed specificbinding capacities of 1000-2900 pmol/mg, corresponding to a 50- to 100-foldpurification over the membrane preparations. The yields were 10-20% of the receptororiginally in the cells, resulting in 50-100 µg from a liter of suspension culture.

Two-step affinity chromatography based on engineered tags can thus be usedto purify recombinant AMPA receptors into essential homogeneity.

35

3.3. IDENTIFICATION OF THE GLUR LIGAND BINDING SITE (II AND III )

When this study was initiated, the membrane topology of glutamate receptorsubunits was unsolved and very controversial. In particular, this concerned the locationof the loop segment between M3 and M4. This loop (S2) had been suggested toparticipate, together with another segment (S1) in the ligand binding (Stern-Bach et al.,1994). On the other hand, the extracellular location of the segment was questioned bystudies identifying phosphorylation sites in this segment (Wang et al., 1993; Raymondet al., 1993; Yakel et al., 1995). To identify whether this loop together with S1 actuallyparticipates in ligand binding, a clearly extracellular event, a dissection of two subunits,GluRD and GluR6, representing two subtypes, was carried out (II and III ).

We designed a series of constructs of the proposed extracellular portions ofthe AMPA receptor subunit GluRD

flip (II , Fig. 1a). These were the N-terminal domain

down to the first transmembrane segment (D/XS1, amino acids 22-546), the first andsecond bacterial periplasmic binding proteins (PBPs) homology segments (D/S1, aa404-546 and D/S2, aa 649-813) both separately and as a fusion protein (D/S1S2). TheN-terminus for S1 was determined from sequence alignments with LAOBP, HisJ andQBP (alignment in Sun et al., 1998; structures in Kang et al., 1991 for LAOBP; Oh etal., 1994 for HisJ; Sun et al., 1998 for QBP). The C-terminus of S1 and the N-terminusof S2 correspond to borders with membrane-associated segments. To obtain a fusionprotein of the S1 and S2 segments of GluRD, we included a hydrophilic linker toprovide flexibility and to possibly aid in folding. This linker is a 12 amino acid peptidefrom the membrane-bound form of IgM in which it connects the Cµ4 domain of theimmunoglobulin heavy chain to the hydrophobic transmembrane domain (Rogers etal., 1980). To the N-terminus we included a GluRD signal sequence for secretion intothe culture medium and a FLAG tag, and to the C-terminus a c-myc-epitope foridentification and purification purposes. The receptor fragments were expressed inHigh Five insect cells, since this cell line secretes proteins efficiently (Davis et al.,1992). The culture media were harvested 3-4 d post-infection and tested for expressionof tagged constructs by immunoblotting. The XS1 fragment was secreted into theculture medium as a 66 kDa band and was detected in the supernatant (II , Fig. 1B).This is slightly higher than the molecular mass expected from the sequence (60 kDa)and is probably due to the addition of N-glycans. The S1 (electrophoretic size 22/expected size 17 kDa) and S1S2 (42/39 kDa) fragments were similarly secreted. TheS2 fragment (22/20 kDa), in contrast, was poorly secreted into the culture media andaccordingly, only a weak myc-immunoreactive band was observed in the supernatant.The positive supernatants were subjected to high-speed centrifugation (160 000 × g)to remove insoluble material. After extensive dialysis of the conditioned culture mediato remove all glutamate present, the samples were tested for radioligand binding. TheXS1 and separate S1 and S2 proteins were negative for [3H]AMPA-binding, as was acontrol supernatant of cells infected with wild type viruses, but the S1S2 constructshowed consistent binding 10-40 times over the background.

The soluble S1S2 construct was purified in one step by using immunoaffinitychromatography. Culture supernatant was passed through an anti-FLAG-tag antibody-Sepharose column and the bound proteins were eluted with EDTA. A western blot

36

analysis revealed a single 42 kDa species detected by monoclonal antibodiesrecognising the FLAG and c-myc epitope tags. In a typical purification, the specificbinding capacity was 6-12 nmol/mg. The highest binding capacity obtained, 12.8nmol/mg, corresponds to ~50% of the theoretical maximum, assuming one bindingsite per S1S2 molecule.

The ligand-binding pharmacology of S1S2 was characterised in detail by usingdisplacement assays and compared to GluRD

flip. The purified S1S2 bound [3H]AMPA

with high affinity (Kd = 50 nM) and competing ligands displaced this binding with a

profile typical for AMPA receptors (Honore et al., 1988; Keinänen et al., 1990). Therank order of potencies was quisqualate (IC

50= 33 nM) >L-glutamate (IC

50= 0.38 µM)

≅DNQX (IC50

= 0.46 µM) >kainate (IC50

= 19 µM). NMDA (100µM) did not affect[3H]AMPA binding of D/S1S2. These values are close to the values obtained forintact GluRD (II , Fig 3b). Thus, D/S1S2 reproduces the binding characteristics ofmembrane-bound GluRD and both S1 and S2 are needed for high-affinity agonist andantagonist binding.

To further confirm the results and to study if the ability of the S1 and S2domains to form a ligand binding site independent of the rest of the protein is a generalproperty of glutamate receptors, a similar ligand-binding fragment was constructedfor the kainate receptor GluR6 (III ). A fragment, 6/S1S2 (amino acids 407-561/662-819), was constructed with the same molecular characteristics as D/S1S2. Thus it hadan EGT (AcNPV ecdysone-S-glycotransferase) signal sequence, the same linkersegment as in D/S1S2 and an N-terminal FLAG tag. In the C-terminus a His tag wasincluded. The construct was produced in High Five insect cells as a secreted proteinwith an electrophoretic size of 43-54 kDa. The culture supernatant was centrifugedand dialysed extensively to remove endogenous glutamate and tested for [3H]kainatebinding. No specific binding was, however, observed.

In parallel, GluR6 fragments, which retained some of the membrane associatedsegments, were analysed. Two deletion mutants were constructed, one which lacksthe X-domain and the segment C-terminal from S2 (S1[M1-M3]S2, aa 407-819) andanother, which lacks the N-terminus until S1 and the membrane domains M1-M3, butcontains an intact C-terminus from S2 fragment (S1S2-M4, aa 407-561/662-908; III ,Fig. 3a). These were expressed in High Five cells, the cells were harvested 3 days posttransfection and examined for expression of recombinant constructs byimmunoblotting. Diffuse or multiple bands with a size range of 48-60 kDa (S1[M1-M3]S2) and 54-60 kDa (S1S2-M4) were observed. The membrane fraction was isolatedand studied for kainate binding. These membranes consistently bound [3H]kainateand the binding was displaced with 1 mM L-glutamate. The ligand bindingcharacteristics of S1[M1-M3]S2 were characterised further. This membrane-boundfragment bound [3H]kainate with high affinity (K

d = 15 nM). The binding was displaced

by domoic acid (Ki = 6 nM), L-glutamate (K

i = 0.8 µM) and CNQX (K

i = 1.2 µM).

100 µM AMPA or L-glutamate did not affect binding. These characteristics weresimilar to intact GluR6.

Since the membrane anchors in the two fragments (S1[M1-M3]S2 and S1S2-M4) were different, but both were able to render the fragments functional in terms ofhigh-affinity [3H]kainate binding, a possibility emerged that anchorage to a support

37

may be needed for functionality. Therefore, the kainate-binding assay was modifiedso that the S1S2 fragment was first bound to a Ni2+ chelating Sepharose matrix andthen incubated with the radioligand and subjected to a filtration assay. Using themodified assay, GluR6/S1S2 exhibited consistently specific [3H]kainate bindingactivity. It did not bind [3H]AMPA, nor did the Sepharose beads without 6/S1S2 orbeads with D/S1S2 bind any [3H]kainate. In spite of the specificity, however,[3H]kainate binding was of relatively low affinity (K

d = 300-400 nM), although accurate

values were difficult to obtain because the binding did not saturate at the radioligandconcentrations up to 300nM. In a competition assay, domoate displaced [3H]kainatewith IC

50 value of 0.33 µM; L-glutamate inhibited binding with an IC

50 value of 15

µM and CNQX with IC50

of 4.5 µM. Compared to intact GluR6, the affinities for theagonists kainate, domoate and glutamate were ~20-fold lower, but the affinity towardsthe antagonist CNQX was similar.

These results show that S1 and S2 form the ligand-binding site in GluR6 andthat the binding site can be expressed as a S1S2 fusion protein in insect cells. Theligand-binding properties of GluR6, however, are not as closely reproduced by theS1S2 fragment as those of GluRD. In 6/S1S2 the affinity for agonists is reduced,whilst the affinity for the antagonist CNQX is similar to membrane-bound GluR6.Binding of [3H]kainate can be measured in the presence of an artificial support, butaffinity is still lower than in the intact GluR6.

3.4. DETERMINANTS FOR LIGAND SELECTIVITY (III )

The two ligand-binding domains from different subtypes of the glutamatereceptors, GluRD/S1S2 and GluR6/S1S2, bind their respective ligands specifically,i.e. D/S1S2 binds [3H]AMPA but not [3H]kainate and 6/S1S2 binds [3H]kainate butnot [3H]AMPA. The structural determinants for ligand selectivity between AMPAand kainate receptors must therefore lie within the ~300 amino acids in the segmentsS1 and S2.

The relative roles of the S1 and S2 segments in ligand selectivity were studiedwith soluble chimaeric ligand binding domains. The chimaeras were generated betweenan AMPA selective GluRD/S1S2 and a kainate selective GluR6/S1S2 (III , Fig. 6).The chimaeric S1S2 fragments were expressed in High Five insect cell cultures, andwere secreted into the culture medium. The culture supernatants were harvested 3-4days post-infection and examined for radioligand binding. Binding of [3H]AMPAwas assayed from samples dialysed against the binding buffer. [3H]kainate bindingactivity was determined with a modified ligand binding assay described above.

First, intact S1 and S2 segments were exchanged. The construct having theS1 segment from GluR6 and the S2 segment from GluRD (6/S1:D/S2) bound both[3H]AMPA and [3H]kainate in a single-point radioligand binding assay, whereas thereverse chimaera D/S1:6/S2 bound neither of the radioligands. Second, furtherchimaeras within the S2 segment were constructed. A 61 amino acid segment at theN-terminus of 6/S2 was replaced with the corresponding part of D/S2. This was joinedwith either D/S1 or 6/S1. The chimaera D/S1:D6/S2 bound [3H]AMPA, but not[3H]kainate. In contrast, the chimaera 6/S1:D6/S2, where the only part derived from

38

GluRD is the 61 amino acid segment in S2, bound both radioligands. A furtherminimisation of the contribution of GluRD by replacing only 32 residues at the N-terminus of S2 in 6/S1S2 resulted in protein that was secreted, but did not have anyligand binding activity. The chimaera 6/S1:D6/S2 was studied in more detail by usingsaturation-binding and ligand-competition assays. The affinity for AMPA was toolow to be measured accurately, with a K

d value > 200 nM. L-glutamate, kainate and

CNQX displaced [3H]AMPA with IC50

values of 0.07 µM, 0.37 µM and 1.3 µM,respectively. The affinities for AMPA and CNQX were lower than in GluRD/S1S2,but the affinities for glutamate and kainate were higher than in GluRD/S1S2.

The results suggest that the N-terminal one-third of S2 contains some importantresidues for selective AMPA binding, as it alone is able to confer AMPA bindingability to 6/S1S2. The chimaeras were all secreted and water soluble, since theyremained in solution upon centrifugation at 160 000 × g. In the construct D/S1:6/S2there is some structural incompatibility that does not allow the binding of ligands tothe binding pocket.

3.5. BIOCHEMICAL CHARACTERISATION OF THE ECTODOMAIN (IV )

Encouraged by the successful production of the ligand-binding domain as aseparate S1S2 fusion protein, we extended this approach to the whole ectodomain andto the N-terminal ~400 amino acids. Importantly, not much is known of the structureand function of the N-terminal domain (X domain) and therefore, this domain is ofspecial interest.

First, we investigated whether the ectodomain and the X domain could beproduced as soluble, secreted proteins. This would facilitate their more detailedanalysis, and eventually, would serve as the first step towards their crystallisation.

Recombinant GluRD constructs were designed to express the extracellulardomains as affinity-tagged proteins. The N-terminal part of the receptor up until theM1 segment (XS1, amino acids 22-546), the first 381 residues (X, aa 22-403), theS1S2 domain harboring the S1 segment before M1 and the loop between M3 and M4,and the whole extracellular part (XS1S2, aa 22-546/649-813) were expressed in HighFive insect cells as immunoreactive proteins of expected sizes and were secreted intothe culture medium (IV , Figs. 1 and 2). The fragments were purified from the culturesupernatants using metal chelation chromatography. In silver stained protein gelsone major band was seen, with no contaminating protein bands. The XS1 fragmentwas the most susceptible one of the fragments to degradation, with proteolytic productsappearing after some weeks of storage on ice. The yields were 100-400 µg of purifiedXS1S2, XS1 and X and 1-2 mg of S1S2 from one-liter cultures.

The purified proteins were analysed for binding of [3H]AMPA and L-[3H]glutamate in a filtration assay. As shown earlier (Stern-Bach et al., 1994; II andIII ), the segments S1 and S2 are necessary for high-affinity ligand binding. Consistentwith this, XS1S2 and S1S2 fragments exhibited specific binding of the radioligands,whilst XS1 and X did not. In a saturation binding assay, the purified XS1S2 boundAMPA with a K

d value (42nM) very similar to that of S1S2 (K

d =33nM) and intact

39

GluRD (Kd = 58nM). In ligand displacement assays, AMPA binding was displaced by

L-glutamate (IC50

= 0.28µM), kainate (IC50

= 2.1µM) and DNQX (IC50

= 0.23µM) withIC

50 values similar to those obtained for S1S2 (respective IC

50 values were 0.29µM,

2.3µM and 0.46µM) and intact GluRD (respective IC50

values were 0.45µM, 3.1µMand 0.58µM; IV , Table I). The filtration assay is sensitive to the ligand off-rate and toobtain more accurate binding parameters and to establish the kinetics of binding,fluorescence-based ligand binding measurements were done. These are based on thequenching of tryptophan fluorescence upon conformational changes induced by bindingof the ligand. The quenching effect can be titrated with ligand to obtain bindingaffinities. The S1S2 and XS1S2 show similar affinities for L-glutamate, with K

d values

of 0.55 µM and 0.49 µM, respectively. These values are somewhat higher, but similar,to the ones obtained from filtration assay (0.29µM for S1S2 and 0.28µM for XS1S2).

Finally, the time course of glutamate-induced fluorescence changes wasfollowed by using stopped-flow rapid-mixing techniques. Under pseudo-first orderconditions ([L]>>[R]), the observed rate constant exhibited linear dependence onglutamate concentration. The association and dissociation rate constants showed nosignificant difference for S1S2 and XS1S2, with k

on= 16 × 106 M-1s-1, k

off= 8 s-1 for

S1S2 and kon

= 15 × 106 M-1s-1, koff

= 6 s-1 for XS1S2 (IV , Table II).Because XS1S2 is able to bind AMPA and L-glutamate with high affinity, the

structure of the X domain in it is probably close to native. For the X domain expressedwithout S1S2 there are no functional assays. Therefore, the integrity of its structurehas to be studied by other means. A recently isolated monoclonal antibody, Fab21,binds to an epitope within the X domain in a conformation-specific manner (Jespersenet al., 2000). Immunoprecipitations with Fab21, were carried out for the solublefragments. All of the fragments containing the X domain (XS1S2, XS1 and X) wereimmunoprecipitated by Fab21, but S1S2 was not. As a control, the X domain washeated briefly at 65oC and denatured. This was not immunoprecipitated by Fab21,thereby verifying the conformation-specificity of the antibody. Thus, the X domainseems to fold correctly when produced as a secreted, soluble construct.

To study the oligomeric state of the fragments, their native molecular weightswere determined. These can be calculated from the Svedberg equation (Eq. 1) if thediffusion (D) and sedimentation constants (s) are known. These hydrodynamicparameters can be determined from size exclusion chromatography (àD) and sucrosedensity gradient centrifugation (às),

sRT M= (Equation 1)

D(1-υρ)

where s is the sedimentation coefficient, R is the gas constant (8.314 JK-1 mol-1), T isthe absolute temperature (293 K), D is the diffusion coefficient, υ is partial specificvolume estimated from the amino acid sequence and ρ is the density of solvent (forwater ρ= 0.9982 g cm-3).

In size exclusion chromatography, X and S1S2 eluted as single symmetric

40

peaks, whereas XS1S2 exhibited a broader peak with a shoulder. XS1 eluted in thevoid volume suggesting that it was aggregated and there was also a substantial loss ofprotein with XS1 suggesting adsorbtion to the gel matrix. Therefore, it was not analysedfurther. The peak fractions were assayed for radioligand binding and aliquots wererun on SDS-PAGE with subsequent immunoblotting. The silver stained and FLAGimmunoreactive receptor bands coincided with the peak fractions of A

280 indicating

that these fractions correspond to the recombinant fragments. The diffusion coefficientswere obtained from a calibration graph in which the partition coefficients (derivedfrom elution volumes) of standard proteins were plotted as a function of the inverse ofdiffusion coefficients, obtained from the literature (Sober, 1970).

In sucrose gradient centrifugation, S1S2 sedimented in a rather narrow band,and X and XS1S2 in slightly more broad bands. The sedimentation coefficicientswere obtained from a calibration graph in which the distance of sedimentation ofstandard proteins was plotted against the sedimentation coefficients found in theliterature. The values of D and s were used with the Svedberg equation to obtain thefollowing results:

The calculated sizes were derived from the amino acid sequences and do notinclude glycans. The native molecular weights suggest that S1S2 is a monomer, whereasX and XS1S2 are dimers in solution. The similarity between the experimental andcalculated size of S1S2 is very close and speaks for the general reliability of themethod.

To further analyse the oligomerisation states of X and XS1S2, cross-linkingexperiments were carried out. Covalent cross-linking was achieved with glutaraldehyde,which converted XS1S2 in a time- and concentration dependent manner into a largermolecular weight complex (Mw around 160-200 kDa). Under the same conditions,S1S2 remained exclusively monomeric (Mw = 42 kDa) and X produced a weak 100kDa band in addition to the major 50 kDa band. These findings are consistent with themonomeric and dimeric structures of S1S2 and XS1S2, respectively, as indicated bythe above hydrodynamic analysis. The dimerisation of the X fragment is, however,more difficult to reconcile with the low level of cross-linked product. In SDS-PAGE,after sucrose gradient centrifugation, the cross-linked XS1S2 was resolved into threepopulations: monomers, dimers (majority) and a minority of much larger aggregatesthat did not penetrate into the running gel (IV , Fig. 7B). As the cross-linked XS1S2fragment sedimentated as a similar broad band as before glutaraldehyde treatment,the cross-linking did not change its properties and thus it can be concluded that thecross-links are intramolecular and not intermolecular. The dimer size band (160-200kDa) constitutes the major population, hence proving that the XS1S2 exists in solution

D(×107 cm2 s-1)

s(×10-13)

Native mol.mass(kDa)

Calculated size(kDa)

S1S2 6.85 3.0 41 38X 5.65 5.3 86 46

XS1S2 3.61 6.7 169 82

41

largely as dimers.From these experiments it can be concluded, that it is feasible to produce

fragments of the extracellular parts of glutamate receptors as separate, soluble domainsin a structurally-relevant native-like state. In addition, the hydrodynamic sizemeasurements indicate that the separately expressed ectodomain exists as a dimer,and that the dimerisation is determined by the presence of the X domain.

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4. DISCUSSION

4.1. PURIFICATION OF A MEMBRANE BOUND CHANNEL PROTEIN

The glutamate receptors, unlike the nicotinic acetylcholine receptors, are notabundant in any native tissue. Native glutamate receptors have, however, beensolubilised, purified and preliminarily characterised from various sources such aspig, chick and rat brain (Chang et al., 1991; Henley and Barnard, 1989; Bahr et al.,1992). The small amounts of receptor obtained from these sources have provedinsufficient for any detailed biochemical or particularly biophysical studies, whichtypically require milligram amounts of highly homogeneous protein. Furthermore,the glutamate receptors in nervous tissue are mostly heteromeric and often associatedwith other proteins, which further complicates their molecular characterisation.Heterologous expression systems provide the possibility to overexpress recombinantreceptors with defined composition for structural analysis.

Glutamate receptors have been expressed in Xenopus oocytes (e.g. Hollmannet al., 1989), in transiently (Keinänen et al., 1990) and stably (Tygesen et al., 1994;Andersen et al., 1996) transfected mammalian cells and in yeast cells (Becker et al.,1998). Apart from yeast, these systems are not suitable for the large-scale biochemicalanalysis of the receptor. In contrast, insect cells have proved easy to cultivate in large-scale suspension cultures in relatively inexpensive serum-free media. Importantly,insect cells are also able to express mammalian proteins with post-translationalmodifications such as N-glycosylation, although differences in the glycans have beenreported (Ogonah et al., 1996). Most commonly, infection by recombinant baculovirusis used to make the insect cells produce heterologous proteins.

In this study, homomeric GluRD AMPA receptors and GluR6 kainate receptorswere produced in Sf21 insect cells, and a purification method for the GluRD receptorswas established. The receptors were solubilised with a nonionic detergent Triton X-100, which solubilised 40-60% of the homomeric GluRD receptors. No changes wereobserved in the ligand binding properties of GluRD upon solubilisation. It has beenreported that solubilisation converts the low affinity pool of AMPA receptors into ahigh affinity state (Hall et al., 1992), but in the present study the solubilised GluRDreceptors exhibited a similar affinity to [3H]AMPA as did the membrane bound GluRD.

Native glutamate receptors solubilised from brain tissue have been partiallypurified by using various chromatographic methods such as ion exchange, lectin- andhydroxyapatite chromatography (Chang et al., 1991; Hunter and Wenthold, 1992).The optimal purification method would use affinity chromatography with a specificligand bound to a column to ensure that only active material would be recovered.However, for glutamate receptors no such ligands are available. We decided to useaffinity tags engineered into the receptor subunits to facilitate the use of a similarpurification protocol for all recombinant receptors (I ). A 5×His tag was engineered atthe carboxy-terminus and a FLAG tag at the amino-terminus of GluRD. These tagsdid not affect the expression or solubilisation properties as compared to the wild typereceptors characterised earlier (Keinänen et al., 1994). The first purification step usesimmobilised metal chelation affinity chromatography and the second step

43

immunoaffinity chromatography. Yields were typically 50-100 µg from a litre of insectcell culture (2x106 cells). A purification of 50 to 100-fold was achieved with a specificactivity of 2000 pmoles of active receptor/mg of protein obtained in a successfulpurification. Both the yield and specific activity of the purified receptors comparefavourably to values reported in the literature, including 370 pmol/mg of AMPA receptorsfrom bovine brain (Hunter and Wenthold, 1992) and 53 pmol/mg AMPA receptorsfrom rat brain (Bahr et al., 1992).

The ligand binding properties of the purified receptor were not different fromthe membrane-bound or solubilised receptor. The best preparations had a specificbinding capacity of 2000 pmol/mg, which represents 20% of the theoretical maximum,assuming one binding site/100kDa subunit. This can be interpreted so that 80 % of thereceptors are inactive or that in multimers only part of the subunits diplay high-affinitybinding. In addition, the determination of B

max may not be accurate, because in a

filtration binding assay the binding sites may not be recovered quantitatively. KSCN,a chaotropic ion, was included in the binding-assay buffer as it had been reported toconvert AMPA receptors into a high-affinity state (Honore and Drejer, 1988). However,if some receptors remained in the low-affinity state, they would not be resolved in thefiltration assay due to the fast dissociation rate.

To further purify the material, the purified receptors can be subjected to size-based separation methods. We have used sucrose density gradient centrifugation andgel filtration to separate aggregates and monomers from the desired single particlesof assembled receptors (IV, and unpublished work). GluRD and GluRB receptorpreparations treated this way have been found to be relatively homogeneous in anelectron microscopical analysis (Dr. Dean Madden, Max Planck Institute for MedicalResearch, Heidelberg). In gel filtration the purified GluRD particles had an apparentmolecular weight of 550 kDa as a detergent complex (IV ). As the amount of detergentbound to the receptor was unknown, the size is consistent with a tetra- or pentamericassembly.

Due to the inherent difficulty in crystallising membrane proteins, only abouta dozen structures are available for them. Following the first determination of a crystalstructure of an integral membrane protein, the photosynthetic reaction centre ofRhodopseudomonas viridis (Michel, 1982), several X-ray and a few electroncrystallographic structures of membrane proteins have been obtained at higher than3.5Å resolution (reviewed by Sakai and Tsukihara, 1998). Generally, those membraneproteins for which the structure has been solved are abundant in nature or they can beproduced on a large scale in bacteria. Remarkably, the structure of a bacterial K+-channel, KcsA, which is similar to the pore domain of a eucaryotic potassium channel,has been solved (Doyle et al., 1998). No crystal structures are yet available for anyneurotransmitter receptors, but a reasonably high resolution structure (4.6Å) has beenobtained by electron microscopy for the acetylcholine receptor (Miyazawa, 1999).

4.2. THE LIGAND BINDING SITE AS AN INDEPENDENT FRAGMENT

In 1994, when the present study was initiated, the membrane topology ofGluR subunits was controversial. In AMPAR subunits there are two discontinuous

44

segments that resemble the bacterial periplasmic polar amino acid –binding proteins(Nakanishi et al., 1990). These segments, S1 and S2, are located before the first putativetransmembrane segment and between the third and fourth segment, respectively.Phosphorylation was reported in the S2 segment at residue Ser684 in GluR6 andSer627 in GluRA suggesting that S1 and S2 were on opposite sides of the membrane(Wang et al., 1993; Raymond et al., 1993; Yakel et al., 1995). On the other hand,glycosylation was reported at GluR6 residue Asn720 suggesting that there could be afifth transmembrane domain in the middle of S2 segment (Roche et al., 1994; Tavernaet al., 1994). However, several studies indicated that the second transmembrane domainM2 has a loop structure (Wo and Oswald, 1994; Hollmann et al., 1994; Bennet andDingledine, 1995); this places the S1 and S2 segments both extracellularly, providedthat M3 is a true transmembrane segment. A study of chimaeric receptors identifiedthe essential role of the S1 and S2 regions in GluRC and GluR6 in the determinationof the ligand pharmacology (Stern-Bach et al., 1994). To solve the controversy intopology and to provide definitive proof for the role of S1 and S2 segments in ligandbinding, a set of soluble constructs of an AMPA receptor GluRD was designed.

Constructs of different segments of the suspected extracellular parts weremade, including the N-terminal domain XS1, the PBP homology domains S1 and S2separately and as a fusion protein joined together with a hydrophilic linker, S1S2. TheS1S2 fusion protein was secreted into the insect cell culture medium, bound [3H]AMPAwith a high affinity (K

d=50 nM) and reproduced the binding characteristics typical for

an AMPA receptor. This demonstrates that the structures needed for high affinityligand-binding lie within the S1 and S2 segments, and since separate S1 and S2 segmentswere inactive, they are both necessary for this property. This result also indicates thatin order to form a ligand binding domain, the S1 and S2 segments must lie on thesame, extracellular side of the membrane. The separately expressed S2 segment wassecreted poorly for unknown reasons. We do not believe that the membrane-associatedsegments within S2 could be the cause for the poor secretion since the whole S1S2construct is secreted efficiently.

Moreover, we also found that the linker peptide between the S1 and S2segments is not critical for high-affinity ligand binding, as a version with a minimallinker of just two amino acids was found to behave similarly to the original S1S2construct (Keinänen et al., 1997).

To study the structural determinants of ligand binding in another GluR subtype,the minimal ligand-binding domains of a kainate receptor GluR6 were constructed(S1[M1-M3]S2, S1S2-M4 and S1S2) and expressed in High Five insect cells. Theintact GluR6 exhibits high-affinity [3H]kainate binding, but somewhat surprisingly, 6/S1S2 did not exhibit any specific [3H]kainate binding. However, the two membraneanchored constructs, S1[M1-M3]S2 and S1S2-M4, exhibited high affinity kainate bindingthat was displaced with L-glutamate. These observations suggested that a solid supportcould be important for the detection of ligand binding activity, accordingly Sepharosebeads were used as a substitute for the membrane anchor. With a modified bindingassay using Sepharose-bound S1S2-samples it was possible to detect specific bindingof [3H]kainate. The binding did not saturate, however, making it difficult to determinethe accurate affinity of kainate-binding. The binding of all agonists (kainate, L-glutamate

45

and domoate) was of relatively low affinity, but the affinity for the antagonist CNQXwas not greatly altered compared to membrane-bound GluR6. This shows that agonistsand antagonists require different conformations for their binding and the agonist affinitywas affected more severely in the GluR6/S1S2 fusion protein.

A similar minimal ligand-binding domain was later constructed for the glycine-binding domain of the NR1 subunit and expressed in insect cells. However, it showedlower affinities for all of the measured agonists as compared to the membrane-boundNR1 receptors (Ivanovic et al., 1998). In contrast, a construct comprising the entireextracellular domain of NR1, showed ligand-binding properties comparable to theintact homomeric NR1 receptors (Miyazaki et al., 1999).

4.3. DETERMINANTS FOR LIGAND SELECTIVITY

GluR6 and GluRD belong to different GluR families and do not co-assembleinto native glutamate receptors (Partin et al., 1993; Puchalski et al., 1994). They share~40% overall amino acid identity, but show different ligand selectivities. AMPA receptorsshow a rank order of agonist potency: quisqualate> AMPA> domoate> L-glutamate>kainate. Conversely, the rank order of agonist potencies at the kainate receptors isdomoate> kainate> quisqualate> L-glutamate.

Chimaeric ligand binding domains of these two subunits were constructed inorder to study the relative roles of S1 and S2 in ligand recognition and to locate thedeterminants responsible for different agonist selectivities. For [3H]AMPA-bindingthe origin of the S1 segment was not critical, but the GluRD N-terminal third of theS2 segment (residues 649–709 in GluRD) was needed, suggesting that some criticalresidues for recognition of agonist AMPA lie within this segment. [3H]Kainate-binding,on the other hand, was dependent on the 6/S1 segment suggesting that the segmentharbors relevant residues for the specificity towards kainate that are not present in D/S1. These results are consistent with the findings on chimaeras made betweenmembrane-bound GluRC and GluR6 subunits (Stern-Bach et al., 1994).

Point mutations have also been studied in order to find out the determinantsfor ligand recognition (reviewed in Dingledine et al., 1999). In the N-terminal regionof S2, which in this study was found to be critical for ligand binding, two residues(L646 and S650) in AMPA receptor GluRA were found to decrease desensitisation(Mano et al., 1996). The crystal structure of GluRB S1S2 revealed the presence ofthree residues (G653, S654 T655) in the N-terminal part of S2 that are critical forinteractions with ligand (Armstrong et al., 1998).

4.4. BIOCHEMICAL ANALYSIS OF OTHER EXTRACELLULAR DOMAINS

To analyse the properties of extracellular domains and to locate determinantsinvolved in the oligomerisation of iGluRs, we studied soluble fragments of GluRDextracellular segments. Two previously uncharacterised protein domains wereconstructed: the whole ectodomain (XS1S2) and the first ~400 residues (X). Forcomparison, S1S2 and the N-terminal stretch before the first transmembrane domain(XS1) were included in the studies. In a suspension culture of High Five insect cells,

46

each construct was secreted into the culture medium. Purification of these receptorfragments was carried out in a one-step metal chelation chromatography with the useof a His-tag engineered at the C-termini of the constructs. The yields were between0.1-2 mg from one liter of culture.

The sequence similarity with the bacterial leucine/isoleucine/-valine-bindingprotein (LIVBP) suggested a role for the X domain in ligand binding. To address thisissue, ligand-binding properties of XS1S2 and S1S2 were compared by filtration assayand fluorescence-based titration and stopped-flow kinetic measurements. These showedthat the affinities for the agonists AMPA, glutamate and kainate, as well as for theantagonist DNQX, are comparable in XS1S2 and S1S2. Thus, the presence of X doesnot impart any change in the ligand-binding event. This is consistent with an earlierstudy showing that swapping of the X segments did not alter the ligand binding propertiesof the GluRC-GluR6 chimaeras compared to parental proteins (Stern-Bach et al., 1994).

How do we know that the purified X domain is folded close to its native structureas there is no functional assay available? First, it is secreted into the culture medium,thus suggesting that it is processed correctly by the cells secretion machinery. Second,XS1S2 exhibits high-affinity ligand binding suggesting that the overall structure mustbe native-like. Furthermore, a monoclonal antibody, Fab21, recognised both intactsolubilised GluRD and GluRD/X in a conformation-specific manner (Jespersen et al.,2000). Taken together, these results strongly suggest that the X fragment is correctlyfolded.

The hydrodynamic size and oligomerisation state of the soluble fragmentswere analysed by calculation of native molecular weights using the diffusion andsedimentation coefficients obtained from gel filtration and sucrose density gradientcentrifugation. Molecular weights of 41 kDa, 86 kDa and 169 kDa were obtained forthe S1S2, X and XS1S2 fragments, respectively. These values are consistent with theX and XS1S2 domains being dimers and the S1S2 domain being a monomer.

The molecules were analysed further by covalent cross-linking of the purifiedfragments by glutaraldehyde, a relatively efficient and non-specific cross-linker. Theectodomain (XS1S2) was cross-linked efficiently, but only a faint 100 kDa-band wasseen with the glutaraldehyde-treated X fragment and no formation of higher molecularweight complexes was observed under the same conditions with S1S2. XS1S2 consistedof mainly a 160-200 kDa species as detected by density gradient centrifugation ofcross-linked XS1S2.

In conclusion, the ligand-binding domain S1S2 behaves as a monomer insolution. This is consistent with recent low-angle X-ray scattering studies, whichindicate a size of 45 kDa for GluRD/S1S2 (Abele et al., 1999). In addition, bacteriallyproduced GluRB/S1S2 has been shown to be a monomer by gel filtration (Chen andGouaux, 1997). The X fragment seems to dimerise spontaneously upon production ininsect cells as does the whole ectodomain XS1S2. Oligomerisation of the ligand-binding site does not seem to affect the ligand binding characteristics as XS1S2 isdimeric and S1S2 is monomeric in solution and yet they show identical bindingproperties.

47

At this point, the relevance of the present findings upon the oligomerisation ofthe intact receptor is not entirely clear. It is interesting, however, that since the intactmembrane bound GluRD is at least a tetramer, these ectodomain-dimers may representan intermediate in assembly. Similar events have been suggested to take place with thenicotinic acetylcholine receptors. They are proposed to form first dimers and trimers,which then assemble into pentamers (Saedi et al., 1991).

The ectodomain of AMPA receptors interacts with other synaptic proteins aswas demonstrated with the Narp protein, which induces clustering of AMPA receptors(O’Brien et al., 1999). Expression of the ectodomain and the separate domains thereofas soluble proteins enables the studies of the interactions in more detail, especiallystudies on the interacting parts of the receptor. Furthermore, characterisation of bindingsites for potential modulatory agents should be now feasible.

4.5. CONCLUDING REMARKS

Glutamate receptors of NMDA, AMPA and kainate subtypes mediateexcitatory neurotransmission and are involved in memory formation, learning and indisorders of the nervous system. Despite extensive molecular characterisation, manyfundamental questions relating to their structure and function have remainedunanswered and thus warrant further biochemical analysis.

In this study, two homomeric glutamate receptors, an AMPA-selective GluRDand a kainate-selective GluR6, were expressed in Sf21 insect cell cultures. Membranesof cells expressing the homomeric receptors bound their respective ligands with highaffinity and specificity and maintained the pharmacology typical for receptors describedin mammalian expression systems. Purification of an AMPA type glutamate receptorwas achieved by using a protocol based on affinity tags. A recombinant GluRD subunitwas engineered to carry a FLAG tag on its amino-terminus and a His tag on thecarboxy-terminus. Two affinity chromatography steps were employed, after whichthe receptor was the only component in the preparation based on visual inspection ofthe silver-stained protein gels. Yields were 50-100 µg from a two-liter batch of insectcell suspension culture, and this protocol should be easily extendable to other receptorsubunits. Production of sufficient amounts of homomeric glutamate receptors forbiochemical and structural studies has thus been established.

Dissection of the receptor subunit into functional domains lead to theidentification of the S1 and S2 segments as the ligand binding sites of GluRD andGluR6. The expression of ligand binding domains as soluble fragments opens a wholenew approach to study the molecular basis for ligand binding. A segment conferringAMPA selectivity was located to the S2 segment by use of chimaeric S1S2 proteins.The ectodomain of GluRD, which comprises all of the extracellularly exposed partsof the receptor, was also expressed as a soluble secreted protein. The ectodomain andits N-terminal part, the X domain, were dimers in contrast to the monomeric S1S2.

The present work also demonstrates that intact glutamate receptors and theirsoluble extracellular domains can be produced and purified in milligram amounts,thus facilitating their structural analysis. Recently, a crystal structure of the S1S2fusion protein of GluRB with a bound non-desensitising agonist kainate has been

48

reported (Armstrong et al., 1998). Further structures of the entire extracellular domainand of the N-terminal segment are now also within experimental reach, and should aidin the understanding of the ligand binding event, the basis for allosteric regulation andthe mechanism of the ligand induced opening of the channel.

49

5. ACKNOWLEDGEMENTS

This study has been carried out at VTT Biotechnology (1994-1997) and at theDepartment of Biosciences, Division of Biochemistry, and the Institute of Biotechnology,University of Helsinki (1998-1999), under the supervision of Professor Kari Keinänen.I am grateful to Research Professor Hans Söderlund at VTT and Professor Carl G.Gahmberg as well as Professor Mart Saarma at the University of Helsinki for excellentworking facilities.

My warmest thanks are due to my supervisor, Professor Kari Keinänen, forintroducing me to the most important molecules in the world (the glutamate receptors).He has always had an enthusiastic attitude for research and has also been encouragingin the character-building period of my PhD studies. I thank Professor Heikki Rauvalaand Professor Mark Johnson for critical reading of my thesis and useful comments onit.

Doctors Dean Madden, Markus Safferling and Rupert Abele at the Max PlanckInstitute for Medical Research in Heidelberg and Doctor Lene Jespersen (now atM&E Biotech) and Professor Jan Engberg at the Royal Danish School of Pharmacy inCopenhagen, are acknowledged as our collaborators. I thank warmly Anja Pallas,Tuula Kuurila and Taru Kostiainen for assistance in the laboratory.

I want to thank all the members of the receptor group for relaxed atmosphereand all the fun. Most of all, Milla is thanked for long-time friendship. Annukka, Michel,David, Sarah and Cai, thank you for all the help in the lab! I also want to express mythanks to the members of gene technology group at VTT.

Finally, I want to thank my friends and parents for their support andencouragement. Karin is thanked for letting me borrow her dog also during the writingof this thesis, so that I would not forget to exercise and Tarja for taking me out tomovies and a (small) glass of wine. My parents have provided me with love andfinancial support and are warmly thanked for both.

The financial support by the Academy of Finland is gratefully acknowledged.

Helsinki, February 2nd, 2000,

Arja

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